JP2021518343A - Anti-dengue virus antibody with cross-reactivity to Zika virus and how to use - Google Patents

Abstract

The present disclosure provides anti-dengue virus (anti-DENV) antibodies that are cross-reactive to Zika virus (ZIKV) and methods for making and using them. Anti-DENV antibodies have several uses, including treatment or prevention of ZIKV infection. In addition, the claimed antibodies may further comprise a mutant Fc region containing LALA, KAES, and ACT5 mutations.

Description

Field of Invention The present invention relates to an anti-Zika virus antibody and a method for using the same.

Background Dengue is the most common arthropod-borne viral disease in the world. The virus that causes dengue fever (referred to herein as DENV) can be classified into four different infectious serotypes, such as DENV-1, DENV-2, DENV-3, and DENV-4. Symptoms of dengue infection include fever, myalgia, headache, decreased platelet count, decreased white blood cell count, impaired blood coagulation, bleeding, and vascular leakage, which can cause dengue shock syndrome. When a person is previously infected with dengue and then exposed to dengue virus, antiviral antibodies may promote the uptake of the virus into host cells, increasing the risk of developing severe forms of dengue in the patient. However, severe forms of dengue can also occur during the initial infection.

Despite being the most common arthropod-borne viral disease, to date there are no drugs available to treat dengue fever. Approaches to dengue as a disease have been primarily directed to treatment to prevent and / or relieve symptoms of infection.

Vaccines and antibody therapeutics are currently under development to prevent and treat viral infections. However, antibody-based therapies are not without risk. One such risk is antibody-dependent enhancement (ADE), in which non-neutralizing antiviral antibodies promote viral entry into host cells, resulting in intracellular infectivity. (Expert Rev Anti Infect Ther (2013) 11, 1147-1157 (Non-Patent Document 1)). The most common mechanism of ADE is the interaction of the virus-antibody complex with the Fc receptor (FcR) on the cell surface via the Fc portion of the antibody. Usually mild viral infections are enhanced by ADE, resulting in a life-threatening illness. It has been reported that an anti-DENV antibody having a mutation in the Fc region that prevents binding to FcγR did not enhance DENV infection (WO2010 / 043977 (Patent Document 1)). However, there is still a need for antibody therapeutics that do not increase the risk of antibody-dependent enhancement.

The cross-reactivity of DENV-specific antibodies to Zika virus has been reported in Ref. [JCI Insight. 2017; 2 (8). Doi: 10.1172 / jci.insight.92428 (Non-Patent Document 2)]. It has also been demonstrated that DENV-specific antibodies can enhance deca infections [Nature. 2016; 536 (7614): 48-53. Doi: 10.1038 / nature18938 (Non-Patent Document 3), Nat Immunol. 2016. 17 (9): 1102-8 (Non-Patent Document 4)].

WO2010 / 043977

Expert Rev Anti Infect Ther (2013) 11, 1147-1157 JCI Insight. 2017; 2 (8). Doi: 10.1172 / jci.insight.92428 Nature. 2016; 536 (7614): 48-53. doi: 10.1038 / nature18938 Nat Immunol. 2016; 17 (9): 1102-8

Overview The present invention provides anti-DENV antibodies, polypeptides containing a mutant Fc region, and methods of their use.

(ここでX₁はRまたはEであり、X₂はRまたはFであり、X₃はG、DまたはLである)(SEQ ID NO: 42)を含む、HVR-H3、
(ii)アミノ酸配列：

(ここでX₁はD、SまたはEであり、X₂はDまたはAである)(SEQ ID NO: 45)を含む、HVR-L3、および
(iii)アミノ酸配列：

(ここでX₁はTまたはRであり、X₂はAまたはRである)(SEQ ID NO: 41)を含む、HVR-H2；
(b) (i)アミノ酸配列：SX₁YX₂H(ここでX₁はNまたはYであり、X₂はIまたはMである)(SEQ ID NO: 40)を含む、HVR-H1、
(ii)アミノ酸配列：

(ここでX₁はTまたはRであり、X₂はAまたはRである)(SEQ ID NO: 41)を含む、HVR-H2、および
(iii)アミノ酸配列：

(ここでX₁はRまたはEであり、X₂はRまたはFであり、X₃はG、DまたはLである)(SEQ ID NO: 42)を含む、HVR-H3；
(c) (i)アミノ酸配列：SX₁YX₂H(ここでX₁はNまたはYであり、X₂はIまたはMである)(SEQ ID NO: 40)を含む、HVR-H1、
(ii)アミノ酸配列：

(ここでX₁はTまたはRであり、X₂はAまたはRである)(SEQ ID NO: 41)を含む、HVR-H2、
(iii)アミノ酸配列：

(ここでX₁はRまたはEであり、X₂はRまたはFであり、X₃はG、DまたはLである)(SEQ ID NO: 42)を含む、HVR-H3、
(iv)アミノ酸配列：

(ここでX₁はDまたはEであり、X₂はKまたはQである)(SEQ ID NO: 43)を含む、HVR-L1、
(v)アミノ酸配列：

(ここでX₁はNまたはEであり、X₂はTまたはFである)(SEQ ID NO: 44)を含む、HVR-L2、および
(vi)アミノ酸配列：

(ここでX₁はD、SまたはEであり、X₂はDまたはAである)(SEQ ID NO: 45)を含む、HVR-L3；または
(d) (i)アミノ酸配列：

(ここでX₁はDまたはEであり、X₂はKまたはQである)(SEQ ID NO: 43)を含む、HVR-L1、
(ii)アミノ酸配列：

(ここでX₁はNまたはEであり、X₂はTまたはFである)(SEQ ID NO: 44)を含む、HVR-L2、および
(iii)アミノ酸配列：

(ここでX₁はD、SまたはEであり、X₂はDまたはAである)(SEQ ID NO: 45)を含む、HVR-L3。
いくつかの態様では、本発明の抗体は、(i) SEQ ID NO: 11のアミノ酸配列を含むHVR-H1、(ii) SEQ ID NO: 13のアミノ酸配列を含むHVR-H2、(iii) SEQ ID NO: 16のアミノ酸配列を含むHVR-H3、(iv) SEQ ID NO: 21のアミノ酸配列を含むHVR-L1、(v) SEQ ID NO: 24のアミノ酸配列を含むHVR-L2、および(vi) SEQ ID NO: 27のアミノ酸配列を含むHVR-L3を含む抗体ではない。 In some embodiments, the isolated anti-DENV antibody of the invention binds to the DENVE protein. In some embodiments, the anti-DENV antibody of the invention comprises:
(a) (i) Amino acid sequence:

(Where X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L) (SEQ ID NO: 42), HVR-H3,
(ii) Amino acid sequence:

(Here X ₁ is D, S or E, X ₂ is D or A) (SEQ ID NO: 45), HVR-L3, and
(iii) Amino acid sequence:

(Here X ₁ is T or R and X ₂ is A or R) (SEQ ID NO: 41), HVR-H2;
(b) (i) Amino acid sequence: SX ₁ YX ₂ H (where X ₁ is N or Y and X ₂ is I or M) (SEQ ID NO: 40), HVR-H 1,
(ii) Amino acid sequence:

(Where X ₁ is T or R and X ₂ is A or R) (SEQ ID NO: 41), HVR-H2, and
(iii) Amino acid sequence:

(Where X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L) (SEQ ID NO: 42), HVR-H3;
(c) (i) Amino acid sequence: SX ₁ YX ₂ H (where X ₁ is N or Y and X ₂ is I or M) (SEQ ID NO: 40), HVR-H 1,
(ii) Amino acid sequence:

(Where X ₁ is T or R and X ₂ is A or R) (SEQ ID NO: 41), HVR-H2,
(iii) Amino acid sequence:

(Where X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L) (SEQ ID NO: 42), HVR-H3,
(iv) Amino acid sequence:

(Where X ₁ is D or E and X ₂ is K or Q) (SEQ ID NO: 43), HVR-L1,
(v) Amino acid sequence:

HVR-L2, including (where X ₁ is N or E and X ₂ is T or F) (SEQ ID NO: 44), and
(vi) Amino acid sequence:

(Where X ₁ is D, S or E and X ₂ is D or A) (SEQ ID NO: 45), HVR-L3; or
(d) (i) Amino acid sequence:

(Where X ₁ is D or E and X ₂ is K or Q) (SEQ ID NO: 43), HVR-L1,
(ii) Amino acid sequence:

HVR-L2, including (where X ₁ is N or E and X ₂ is T or F) (SEQ ID NO: 44), and
(iii) Amino acid sequence:

HVR-L3 containing (where X ₁ is D, S or E and X ₂ is D or A) (SEQ ID NO: 45).
In some embodiments, the antibodies of the invention are HVR-H1, which comprises the amino acid sequence of (i) SEQ ID NO: 11, HVR-H2, which comprises the amino acid sequence of (ii) SEQ ID NO: 13, and (iii) SEQ. HVR-H3 containing the amino acid sequence of ID NO: 16, HVR-L1 containing the amino acid sequence of (iv) SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of (v) SEQ ID NO: 24, and (vi) ) SEQ ID NO: Not an antibody containing HVR-L3 containing the amino acid sequence of 27.

In some embodiments, the isolated anti-DENV antibody of the invention comprises:
(a) (i) HVR-H3 from any one VH sequence of SEQ ID NO: 2-6, (ii) HVR-L3 from any one VL sequence of SEQ ID NO: 8-10, and (iii) SEQ ID NO: HVR-H2 derived from any one of the VH sequences 2 to 6;
(b) (i) HVR-H1 from any one VH sequence of SEQ ID NO: 2-6, (ii) HVR-H2 from any one VH sequence of SEQ ID NO: 2-6, and (iii) SEQ ID NO: HVR-H3 derived from any one of the VH sequences 2 to 6;
(c) (i) HVR-H1 derived from any one VH sequence of SEQ ID NO: 2 to 6, (ii) HVR-H2 derived from any one VH sequence of SEQ ID NO: 2 to 6, ( iii) SEQ ID NO: HVR-H3 derived from any one VH sequence of 2 to 6, (iv) SEQ ID NO: HVR-L1 derived from any one of VL sequences of 8 to 10, (v) SEQ ID NO: HVR-L2 from any one VL sequence of 8-10, and (vi) SEQ ID NO: HVR-L3 from any one VL sequence of 8-10;
(d) (i) HVR-L1 from any one VL sequence of SEQ ID NO: 8-10, (ii) HVR-L2 from any one VL sequence of SEQ ID NO: 8-10, and (iii) SEQ ID NO: HVR-L3 from any one of the VL sequences 8-10; or
(e) (i) HVR-H1 derived from any one VH sequence of SEQ ID NO: 2 to 6, (ii) HVR-H2 derived from any one VH sequence of SEQ ID NO: 2 to 6, ( iii) SEQ ID NO: HVR-H3 from any one of the VH sequences 2 to 6, (iv) SEQ ID NO: HVR-L1 from any one of the VL sequences of 7, (v) SEQ ID NO: HVR-L2 from any one VL sequence of 7 and HVR-L3 from any one VL sequence of (vi) SEQ ID NO: 7.
In some embodiments, the antibodies of the invention are (i) HVR-H1 from the VH sequence of SEQ ID NO: 1, (ii) HVR-H2 from the VH sequence of SEQ ID NO: 1, and (iii) SEQ. HVR-H3 from VH sequence with ID NO: 1, (iv) HVR-L1 from VL sequence with SEQ ID NO: 7, (v) HVR-L2 from VL sequence with SEQ ID NO: 7, and (vi) ) SEQ ID NO: 7 is not an antibody containing HVR-L3 derived from the VL sequence.

In some embodiments, the isolated anti-DENV antibody of the invention is FR1, which comprises the amino acid sequence of heavy chain variable domain framework SEQ ID NO: 31, FR2, which comprises the amino acid sequence of SEQ ID NO: 32, SEQ ID. It further comprises FR3 containing the amino acid sequence of NO: 33 or 34, and FR4 containing the amino acid sequence of SEQ ID NO: 35. In some embodiments, the isolated anti-DENV antibody of the invention is FR1, which comprises the amino acid sequence of light chain variable domain framework SEQ ID NO: 36, FR2, which comprises the amino acid sequence of SEQ ID NO: 37, SEQ ID. It further comprises FR3 containing the amino acid sequence of NO: 38 and FR4 containing the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the isolated anti-DENV antibody of the invention has a VH sequence having at least 95% sequence identity to any one of the amino acid sequences of (a) SEQ ID NO: 2-6; (b) VL sequence having at least 95% sequence identity to any one amino acid sequence of SEQ ID NO: 8-10; (c) SEQ ID NO: VH sequence of any one of 2-6 and Contains any one VL sequence of SEQ ID NO: 8-10; or (d) any one VH sequence of SEQ ID NO: 2-6 and any one VL sequence of SEQ ID NO: 7.

In some embodiments, the isolated anti-DENV antibody of the invention is a monoclonal antibody. In some embodiments, the isolated anti-DENV antibody of the invention is a human antibody, humanized antibody, or chimeric antibody. In some embodiments, the isolated anti-DENV antibody of the invention is an antibody fragment that binds to DENV or DENVE protein. In some embodiments, the isolated anti-DENV antibody of the invention is a full-length IgG antibody.

In some embodiments, the Fc region of the anti-DENV antibody of the invention comprises Ala at position 234 and Ala at position 235 according to EU numbering. In some embodiments, the Fc region of the anti-DENV antibody of the invention can be selected from the mutant Fc regions described herein.

The present invention also provides an isolated nucleic acid encoding the anti-DENV antibody of the present invention. The present invention also provides a host cell containing the nucleic acid of the present invention. The present invention also provides a method for producing an antibody, which comprises the step of culturing the host cell of the present invention so that the antibody is produced.

The present invention also provides a pharmaceutical preparation containing the anti-DENV antibody of the present invention and a pharmaceutically acceptable carrier.

The anti-DENV antibody of the present invention may be for use as a pharmaceutical. In some embodiments, the anti-DENV antibody of the invention may be for use in the treatment of DENV infection.

The anti-DENV antibody of the present invention can be used in the manufacture of pharmaceuticals. In some embodiments, the drug is for the treatment of DENV infections.

The present invention also provides a method of treating an individual suffering from a DENV infection. In some embodiments, the method comprises administering to the individual an effective amount of an anti-DENV antibody of the invention. In some embodiments, the method further comprises administering to the individual an additional therapeutic agent, eg, as described below.

In some embodiments, the mutant Fc region of the invention comprises at least one amino acid modification in the parent Fc region. In a further aspect, the mutant Fc region has a substantially reduced FcγR binding activity when compared to the parent Fc region. In a further aspect, the mutant Fc region does not have substantially reduced C1q binding activity when compared to the parent Fc region.

In some embodiments, the mutant Fc region of the invention comprises Ala at position 234 and Ala at position 235 according to EU numbering. In a further aspect, the mutant Fc region comprises an amino acid modification of any one of (a)-(c) below: (a) positions 267, 268, and 324, (b) 236, 267, 268, 324. , And 332, and (c) 326 and 333; follow EU numbering.

In some embodiments, the mutant Fc region of the invention comprises an amino acid selected from the group consisting of: (a) Glu at position 267, (b) Phe at position 268, (c) Thr at position 324. , (D) Ala at 236, (e) Glu at 332, (f) Ala at 326, Asp, Glu, Met or Trp, and (g) Ser at 333; follow EU numbering.

In some embodiments, the mutant Fc region of the invention further comprises an amino acid selected from the group consisting of: (a) Ala at position 434, (b) Ala at position 434, Thr at position 436, 438. Arg and Glu at 440, (c) Leu at 428, Ala at 434, Thr at 436, Arg at 438, and Glu at 440, and (d) Leu at 428, 434. Ala, Arg at 438, and Glu at 440; follow EU numbering.

In some embodiments, the mutant Fc region of the invention comprises any of the amino acid modifications, alone or in combination, as described in Table 4. In some embodiments, the parent Fc region described in the present invention is derived from human IgG1. The present invention provides a polypeptide comprising any one amino acid sequence of SEQ ID NO: 51-59.

In some embodiments, the polypeptide comprising the mutant Fc region of the invention is an antibody. In a further aspect, the antibody is an antiviral antibody. In a further aspect, the antibody comprises a variable region derived from the anti-DENV antibody described herein.

In a further aspect, the antibody comprises:
(a) (i) HVR-H3 containing the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 27, and (iii) the amino acid sequence of SEQ ID NO: 13. Including HVR-H2;
(b) HVR-H1 containing the amino acid sequence of (i) SEQ ID NO: 11, HVR-H2 containing the amino acid sequence of (ii) SEQ ID NO: 13, and (iii) the amino acid sequence of SEQ ID NO: 16. Including HVR-H3;
(c) (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 13, and (iii) containing the amino acid sequence of SEQ ID NO: 16. HVR-H3, HVR-L1 containing the amino acid sequence of (iv) SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of (v) SEQ ID NO: 24, and the amino acid sequence of (vi) SEQ ID NO: 27 Including HVR-L3; or
(d) (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) the amino acid sequence of SEQ ID NO: 27. Including HVR-L3.

In a further aspect, the antibody comprises:
(a) (i) HVR-H3 from the VH sequence of SEQ ID NO: 6, (ii) HVR-L3 from the VL sequence of SEQ ID NO: 10, and (iii) from the VH sequence of SEQ ID NO: 6. HVR-H2;
(b) (i) HVR-H1 from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 from the VH sequence of SEQ ID NO: 6, and (iii) from the VH sequence of SEQ ID NO: 6. HVR-H3;
(c) (i) HVR-H1 derived from VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from VH sequence of SEQ ID NO: 6, (iii) derived from VH sequence of SEQ ID NO: 6. HVR-H3, HVR-L1 from the VL sequence of (iv) SEQ ID NO: 10, HVR-L2 from the VL sequence of (v) SEQ ID NO: 10, and the VL sequence of (vi) SEQ ID NO: 10. Origin HVR-L3;
(d) (i) HVR-L1 from the VL sequence of SEQ ID NO: 10, (ii) HVR-L2 from the VL sequence of SEQ ID NO: 10, and (iii) from the VL sequence of SEQ ID NO: 10. HVR-L3; or
(e) (i) HVR-H1 derived from VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from VH sequence of SEQ ID NO: 6, (iii) derived from VH sequence of SEQ ID NO: 6. HVR-H3, HVR-L1 from the VL sequence of (iv) SEQ ID NO: 7, HVR-L2 from the VL sequence of (v) SEQ ID NO: 7, and the VL sequence of (vi) SEQ ID NO: 7. Origin HVR-L3.

The present invention also provides an isolated nucleic acid encoding a polypeptide containing the mutant Fc region of the present invention. The present invention also provides a host cell containing the nucleic acid of the present invention. The present invention also provides a method for producing a polypeptide, which comprises the step of culturing the host cell of the present invention so that the polypeptide containing the mutant Fc region is produced.

The present invention also provides a pharmaceutical preparation containing a polypeptide containing the mutant Fc region of the present invention and a pharmaceutically acceptable carrier.

The polypeptide containing the mutant Fc region of the present invention may be for use as a pharmaceutical. In some embodiments, the polypeptide comprising the mutant Fc region of the invention may be for use in the treatment of viral infections.

The polypeptide containing the mutant Fc region of the present invention can be used in the manufacture of pharmaceuticals. In some embodiments, the drug is for the treatment of viral infections.

The present invention also provides a method of treating an individual suffering from a viral infection. In some embodiments, the method comprises administering to the individual a polypeptide comprising an effective amount of the mutant Fc region of the invention.

The present invention also provides the anti-DENV antibodies described herein further comprising a polypeptide comprising the mutant Fc region of the present invention.

In one aspect, the invention provides a method of treating or preventing Zika virus infection.

In some embodiments, the method of treating or preventing Zika virus infection of the present invention comprises administering an antibody that binds to Zika virus.

(ここでX₁はRまたはEであり、X₂はRまたはFであり、X₃はG、DまたはLである)(SEQ ID NO: 42)を含む、HVR-H3、
(ii)アミノ酸配列：QQFX₁X₂LPIT(ここでX₁はD、SまたはEであり、X₂はDまたはAである)(SEQ ID NO: 45)を含む、HVR-L3、および
(iii)アミノ酸配列：

(ここでX₁はTまたはRであり、X₂はAまたはRである)(SEQ ID NO: 41)を含む、HVR-H2；
(b) (i)アミノ酸配列：SX₁YX₂H(ここでX₁はNまたはYであり、X₂はIまたはMである)(SEQ ID NO: 40)を含む、HVR-H1、
(ii)アミノ酸配列：

(ここでX₁はTまたはRであり、X₂はAまたはRである)(SEQ ID NO: 41)を含む、HVR-H2、および
(iii)アミノ酸配列：

(ここでX₁はRまたはEであり、X₂はRまたはFであり、X₃はG、DまたはLである)(SEQ ID NO: 42)を含む、HVR-H3；
(c) (i)アミノ酸配列：SX₁YX₂H(ここでX₁はNまたはYであり、X₂はIまたはMである)(SEQ ID NO: 40)を含む、HVR-H1、
(ii)アミノ酸配列：

(ここでX₁はTまたはRであり、X₂はAまたはRである)(SEQ ID NO: 41)を含む、HVR-H2、
(iii)アミノ酸配列：

(ここでX₁はRまたはEであり、X₂はRまたはFであり、X₃はG、DまたはLである)(SEQ ID NO: 42)を含む、HVR-H3、
(iv)アミノ酸配列：

(ここでX₁はDまたはEであり、X₂はKまたはQである)(SEQ ID NO: 43)を含む、HVR-L1、
(v)アミノ酸配列：DASX₁LKX₂(ここでX₁はNまたはEであり、X₂はTまたはFである)(SEQ ID NO: 44)を含む、HVR-L2、および
(vi)アミノ酸配列：QQFX₁X₂LPIT(ここでX₁はD、SまたはEであり、X₂はDまたはAである)(SEQ ID NO: 45)を含む、HVR-L3；または
(d) (i)アミノ酸配列：

(ここでX₁はDまたはEであり、X₂はKまたはQである)(SEQ ID NO: 43)を含む、HVR-L1、
(ii)アミノ酸配列：DASX₁LKX₂(ここでX₁はNまたはEであり、X₂はTまたはFである)(SEQ ID NO: 44)を含む、HVR-L2、および
(iii)アミノ酸配列：QQFX₁X₂LPIT(ここでX₁はD、SまたはEであり、X₂はDまたはAである)(SEQ ID NO: 45)を含む、HVR-L3。いくつかの態様では、本発明の抗体は、(i) SEQ ID NO: 11のアミノ酸配列を含むHVR-H1、(ii) SEQ ID NO: 13のアミノ酸配列を含むHVR-H2、(iii) SEQ ID NO: 16のアミノ酸配列を含むHVR-H3、(iv) SEQ ID NO: 21のアミノ酸配列を含むHVR-L1、(v) SEQ ID NO: 24のアミノ酸配列を含むHVR-L2、および(vi) SEQ ID NO: 27のアミノ酸配列を含むHVR-L3を含む抗体ではない。 In some embodiments, the antibody that binds to the Zika virus of the invention comprises:
(a) (i) Amino acid sequence:

(Where X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L) (SEQ ID NO: 42), HVR-H3,
(ii) Amino acid sequence: _{HVR-L3, including QQFX 1} X ₂ LPIT (where X ₁ is D, S or E, X ₂ is D or A) (SEQ ID NO: 45), and
(iii) Amino acid sequence:

(Here X ₁ is T or R and X ₂ is A or R) (SEQ ID NO: 41), HVR-H2;
(b) (i) Amino acid sequence: SX ₁ YX ₂ H (where X ₁ is N or Y and X ₂ is I or M) (SEQ ID NO: 40), HVR-H 1,
(ii) Amino acid sequence:

(Where X ₁ is T or R and X ₂ is A or R) (SEQ ID NO: 41), HVR-H2, and
(iii) Amino acid sequence:

(Where X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L) (SEQ ID NO: 42), HVR-H3;
(c) (i) Amino acid sequence: SX ₁ YX ₂ H (where X ₁ is N or Y and X ₂ is I or M) (SEQ ID NO: 40), HVR-H 1,
(ii) Amino acid sequence:

(Where X ₁ is T or R and X ₂ is A or R) (SEQ ID NO: 41), HVR-H2,
(iii) Amino acid sequence:

(Where X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L) (SEQ ID NO: 42), HVR-H3,
(iv) Amino acid sequence:

(Where X ₁ is D or E and X ₂ is K or Q) (SEQ ID NO: 43), HVR-L1,
(v) Amino acid sequence: _{HVR-L2, including DASX 1} LKX ₂ (where X ₁ is N or E and X ₂ is T or F) (SEQ ID NO: 44), and
(vi) Amino acid sequence: _{HVR-L3; or containing QQFX 1} X ₂ LPIT (where X ₁ is D, S or E and X ₂ is D or A) (SEQ ID NO: 45).
(d) (i) Amino acid sequence:

(Where X ₁ is D or E and X ₂ is K or Q) (SEQ ID NO: 43), HVR-L1,
(ii) Amino acid sequence: _{HVR-L2, including DASX 1} LKX ₂ (where X ₁ is N or E and X ₂ is T or F) (SEQ ID NO: 44), and
(iii) Amino acid sequence: _{HVR-L3 comprising QQFX 1} X ₂ LPIT (where X ₁ is D, S or E and X ₂ is D or A) (SEQ ID NO: 45). In some embodiments, the antibodies of the invention are HVR-H1, which comprises the amino acid sequence of (i) SEQ ID NO: 11, HVR-H2, which comprises the amino acid sequence of (ii) SEQ ID NO: 13, and (iii) SEQ. HVR-H3 containing the amino acid sequence of ID NO: 16, HVR-L1 containing the amino acid sequence of (iv) SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of (v) SEQ ID NO: 24, and (vi) ) SEQ ID NO: Not an antibody containing HVR-L3 containing the amino acid sequence of 27.

In some embodiments, the antibody that binds to the dicavirus of the invention is a heavy chain variable domain framework FR1 containing the amino acid sequence of SEQ ID NO: 31, FR2 containing the amino acid sequence of SEQ ID NO: 32, SEQ ID NO. : FR3 containing the amino acid sequence of 33 or 34, and FR4 containing the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibody that binds to the dicavirus of the invention is the light chain variable domain framework FR1, which comprises the amino acid sequence of SEQ ID NO: 36, FR2, which comprises the amino acid sequence of SEQ ID NO: 37, SEQ ID NO. It further contains FR3 containing the amino acid sequence of: 38 and FR4 containing the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the antibody that binds to the dicavirus of the invention is a VH sequence having at least 95% sequence identity to any one amino acid sequence of (a) SEQ ID NO: 2-6; b) SEQ ID NO: VL sequence having at least 95% sequence identity to any one amino acid sequence of 8-10; (c) SEQ ID NO: VH sequence of any one of 2-6 and SEQ Includes any one VL sequence with ID NO: 8-10; or (d) SEQ ID NO: VH sequence with any one of 2-6.

The present invention also provides a method of treating a deer infection. In some embodiments, the method comprises administering to an individual an effective amount of an anti-dica antibody of the invention.

In some embodiments, the mutant Fc region of the antibody that binds to the Zika virus of the invention comprises Ala at position 234 and Ala at position 235, according to EU numbering. In a further aspect, the mutant Fc region comprises an amino acid modification of any one of (a)-(c) below: (a) positions 267, 268, and 324, (b) 236, 267, 268, 324. , And 332, and (c) 326 and 333; follow EU numbering.

In some embodiments, the mutant Fc region of the antibody that binds the Zika virus of the invention comprises an amino acid selected from the group consisting of: (a) Glu at position 267, (b) Phe at position 268, (c) Thr at 324, (d) Ala at 236, (e) Glu at 332, (f) Ala at 326, Asp, Glu, Met or Trp, and (g) Ser at 333; EU Follow the numbering.

In some embodiments, the mutant Fc region of the antibody that binds to the Zika virus of the invention further comprises an amino acid selected from the group consisting of: (a) Ala at position 434, (b) Ala at position 434: , 436th Thr, 438th Arg, and 440th Glu, (c) 428th Leu, 434th Ala, 436th Thr, 438th Arg, and 440th Glu, and (d) Follow EU numbering: Leu at 428, Ala at 434, Arg at 438, and Glu at 440.

The present invention provides a polypeptide comprising any one amino acid sequence of SEQ ID NO: 51-59.

In a further aspect, the antibody that binds to the Zika virus of the invention comprises:
(a) (i) HVR-H3 containing the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 27, and (iii) the amino acid sequence of SEQ ID NO: 13. Including HVR-H2;
(b) HVR-H1 containing the amino acid sequence of (i) SEQ ID NO: 11, HVR-H2 containing the amino acid sequence of (ii) SEQ ID NO: 13, and (iii) the amino acid sequence of SEQ ID NO: 16. Including HVR-H3;
(c) (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 13, and (iii) containing the amino acid sequence of SEQ ID NO: 16. HVR-H3, HVR-L1 containing the amino acid sequence of (iv) SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of (v) SEQ ID NO: 24, and the amino acid sequence of (vi) SEQ ID NO: 27 Including HVR-L3; or
(d) (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) the amino acid sequence of SEQ ID NO: 27. Including HVR-L3.

In a further aspect, the antibody that binds the dicavirus of the invention is selected from the group consisting of SG182 (SEQ ID NO: 46), SG1095 (SEQ ID NO: 54), and SG1106 (SEQ ID NO: 59). A VH mutant sequence selected from the group consisting of 3CH1047 (SEQ ID NO: 6) and 3CH1049 (SEQ ID NO: 95) containing an IgG1 CH sequence; and a human CL sequence SK1 (SEQ ID NO: 60). Contains VL mutant sequences selected from the group consisting of 3CL (SEQ ID NO: 7) and 3CL633 (SEQ ID NO: 98).

FIGS. 1 (A)-(D) show DENV-1 E protein (A), DENV-2 E protein (B), DENV-3 E protein (C), and DENV as described in Example 2. -4 BIACORE® sensorgrams of anti-DENV antibodies 3C and 3Cam against E protein (D) are shown. See the description in Figure 1A. See the description in Figure 1A. See the description in Figure 1A. FIG. 2 shows the binding affinity of antibodies with different Fc variants to human C1q, as described in Example 4. Binding activity was measured by ELISA. The Fc mutants tested were WT, LALA + KWES, LALA + EFT + AE, LALA + EFT, and LALA. FIG. 3 shows the binding affinity of antibodies with different Fc variants to human C1q, as described in Example 4. Binding activity was measured by ELISA. The Fc mutants tested were WT, LALA, LALA + KWES, LALA + KAES, LALA + KDES, LALA + KEES, and LALA + KMES. FIG. 4 shows the binding affinity of antibodies with different Fc variants to human C1q, as described in Example 4. Binding activity was measured by ELISA. The Fc mutants tested were WT, LALA, LALA + ACT3, LALA + ACT5, LALA + KAES, LALA + ACT3 + KAES, and LALA + ACT5 + KAES. FIG. 5 shows the binding affinity of antibodies with different Fc variants to mouse C1q, as described in Example 4. Binding activity was measured by ELISA. The Fc mutants tested were WT, LALA, LALA + ACT3, LALA + ACT5, LALA + KAES, LALA + ACT3 + KAES, and LALA + ACT5 + KAES. 6 (a)-(h) show Biacore analysis for Fc mutants that bind to human FcγR, as described in Example 5. The Fc mutants tested were WT (denoted as WT IgG in this figure), KWES, EFT + AE, EFT, KAES, LALA + KWES, LALA + EFT + AE, LALA + EFT, LALA, LALA + ACT3, LALA + ACT5, LALA + KAES, LALA + KAES + ACT3, and LALA + KAES + ACT5. These Fc mutants are (a) human FcγR1a, (b) human FcγR2a allele mutant 167H, (c) human FcγR2a allele mutant 167R, (d) human FcγR2b, (e) human FcγR3a allele mutant 158F, ( FcγR binding was tested, including f) human FcγR3a allelic mutant 158V, (g) human FcγR3b allelic mutant NA1, and (h) human FcγR3b allelic mutant NA2. Each Fc variant is in both the form of an antibody alone (described as Ab alone) carrying the Fc variant and the form of an immune complex (described as CD154 IC) formed of the antibody and the trimer CD154 antigen. evaluated. See the description in Figure 6a. See the description in Figure 6a. See the description in Figure 6a. See the description in Figure 6a. See the description in Figure 6a. See the description in Figure 6a. See the description in Figure 6a. 7 (a)-(d) show Biacore analysis for Fc mutants that bind to mouse FcγR, as described in Example 5. The Fc mutants tested were WT (denoted as WT IgG in this figure), KWES, EFT + AE, EFT, KAES, LALA + KWES, LALA + EFT + AE, LALA + EFT, LALA, LALA + ACT3, LALA + ACT5, LALA + KAES, LALA + KAES + ACT3, and LALA + KAES + ACT5. These Fc variants were tested for binding to FcγR, including (a) mouse FcγR1, (b) mouse FcγR2b, (c) mouse FcγR3, and (d) mouse FcγR4. Each Fc variant is in both the form of an antibody alone (described as Ab alone) carrying the Fc variant and the form of an immune complex (described as CD154 IC) formed of the antibody and the trimer CD154 antigen. evaluated. See the description in Figure 7a. See the description in Figure 7a. See the description in Figure 7a. FIG. 8 shows a Biacore analysis of Fc variants that bind to human FcRn, as described in Example 5. The Fc mutants tested were WT (denoted as hIgG1 in this figure), LALA, LALA + ACT3, LALA + ACT5, LALA + KAES, LALA + KAES + ACT3, and LALA + KAES + ACT5. Each Fc variant is in both the form of an antibody alone (described as Ab alone) carrying the Fc variant and the form of an immune complex (described as CD154 IC) formed of the antibody and the trimer CD154 antigen. evaluated. FIG. 9 shows viremia 3 days after infection of AG129 mice with DENV-2 virus, as described in Example 6. Anti-DENV antibodies 3C and 3Cam in combination with WT (described as hIgG1), LALA, and Fc variants of LALA + KAES, or PBS as a negative control were administered 2 days after virus infection. FIG. 10 shows viremia on the third day after infection of AG129 mice with DENV1-4 virus, as described in Example 6. Anti-DENV antibodies 3C and 3Cam2 with the same Fc mutant (LALA + KAES), or P1 buffer as a negative control, were administered 2 days after virus infection. LOD: Detection limit. Each symbol represents viremia from one mouse. A one-way ANOVA test was used to calculate significant differences between groups. FIG. 11 shows viremia 3 days after infection of AG129 mice with DENV 2 virus, as described in Example 6. Anti-DENV antibodies 3Cam2-LALA and 3Cam2-LALA + KAES, or P1 buffer as a negative control, were administered 2 days after virus infection. 12 (A)-(D) show DENV-1 E protein (A), DENV-2 E protein (B), DENV-3 E protein (C), and DENV as described in Example 2. -4 BIACORE® sensorgrams of anti-DENV antibodies 3C and 3Cam2 against E protein (D) are shown. See description in Figure 12A. See description in Figure 12A. See description in Figure 12A. Figures 13 (A)-(B) show the deca neutralization assay using BHK-21-DC-SIGN cells. The deer strain KF993678 was used. Each data point is from a single measurement. In the first experiment (A), the approximate NT50 of 3Cam2-LALA + KAES + ACT5 is 0.33 μg / mL, and that of 3C-LALA + KAES + ACT5 is 5.33 μg / mL. In the second experiment (B), the approximate NT50 of 3Cam2-LALA + KAES + ACT5 is 0.93 μg / mL. Figures 15 (A)-(B) show the K562 cell ADE assay for Zika virus. The deer strain KF993678 was used. A) Enhancement can be observed in the wild-type version of 3Cam2, but not in its Fc mutants 3Cam2-LALA + KAES and 3Cam2-LALA + KAES + ACT5. INFECTED: No antibody added. The average value ± error (range) of n = 2 is shown. B) Experiments were repeated with a new batch of 3Cam2-LALA + KAES + ACT5, using a wild-type version of 3Cam2 as a control. The average value ± error (range) of n = 2 is shown. FIG. 16 shows the in vivo efficacy against deer infection. Mice were intraperitoneally infected with Zika virus and intravenously treated with 100 μg of 3Cam2-LALA + KAES + ACT5 (3Cam2-SG1106) or 3C-LALA + KAES + ACT5 (3C-SG1106) 2 days after infection. P1 buffer was used as a control. Each dot represents one mouse and the mean ± SD is shown. Kruskal Wallis was used to test the significance; *: p <0.05, **: p <0.005. FIG. 17 shows the survival rate of deer-infected IFNAR mice after antibody treatment. Mice were intraperitoneally infected with Zika virus and treated intravenously with 100 μg of 3Cam2-LALA + KAES + ACT5 or 3C-LALA + KAES + ACT5 2 days after infection. P1 buffer was used as a control. The survival rate per group for 40 days after infection is shown. FIG. 18 shows the binding activity of 3C, 3C-LALA and 3Cam2-LALA + KAES + ACT5 monoclonal antibodies to ZIKV. Binding ELISA curve on purified ZIKV virions. FIG. 19 shows the in vitro neutralizing activity of 3C, 3C-LALA and 3Cam2-LALA + KAES + ACT5 human antibodies against ZIKV. Shows the relative infectivity of antibody-incubated virus compared to virus alone. Two different (old and new) batches of 3C and 3C-LALA were used. Figures 20 (A)-(B) show that antibody treatment prevents ZIKV-induced congenital dysgenesis in IFNαR KO mice. (A) From mock-infected mice (a), ZIKV-infected mice treated with isotype antibody (b), ZIKV-infected mice treated with 3C-LALA (c), and ZIKV-infected mice treated with 3Cam2-LALA + KAES + ACT5 (d). A typical fetal. (B) Fetal weight in each group (n): whole body weight (a) and head weight (b). Each dot represents one foetation. Figures 21 (a)-(d) show that antibody treatment significantly reduces the transmission of ZIKV from mother to foetation. Viral load in amniotic fluid (a), placenta (b), fetal brain (c) and fetal liver (d). Each dot represents one foetation; n ≧ 7 in each group.

Detailed Description of Aspects of the Invention The techniques and procedures described or cited herein are generally well understood, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor. Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al. Eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ) MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); G ene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds) ., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty.,, ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane) (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., Eds., JB Lippincott) Commonly used by those skilled in the art, using conventional techniques such as the widely used techniques described in Company, 1993).

I. Definitions Unless defined otherwise, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1994) , NY 1992) provides those skilled in the art with general guidance for many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

For the purposes of interpreting this specification, the following definitions apply, and whenever applicable, terms used in the singular form also include the plural form and vice versa. It should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. In the event that any of the definitions below conflicts with any document incorporated herein by reference, the definitions below shall prevail.

The term "acceptor human framework" as used herein is derived from the human immunoglobulin framework or human consensus framework defined below, the light chain variable domain (VL) framework or the heavy chain variable domain (VH). ) A framework that contains the amino acid sequence of the framework. Acceptors "derived" from the human immunoglobulin framework or the human consensus framework may include their same amino acid sequence or may include a modification of the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

"Affinity" refers to the total intensity of non-covalent interactions between a molecule (eg, antibody) binding site and a molecule's binding partner (eg, antigen). Unless otherwise indicated, "binding affinity" as used herein refers to a unique binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of the molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific examples and exemplary embodiments for measuring binding affinity are described below.

An "affinity mature" antibody is one or more modifications that result in improved affinity of the antibody for an antigen in one or more hypervariable regions (HVRs) compared to the unmodified parent antibody. It refers to an antibody accompanied by.

The terms "anti-DENV antibody" and "antibody that binds to DENV" refer to an antibody that can bind to DENV with sufficient affinity, so that the antibody is a diagnostic agent and / or treatment when targeting DENV. It is useful as a medicine. The antibody can bind to the E protein of DENV. The terms "anti-DENV E protein antibody" and "antibody that binds to DENV E protein" refer to an antibody that is capable of binding to DENVE protein with sufficient affinity, so that the antibody is intended to target DENV. It is useful as a diagnostic and / or therapeutic agent. In one aspect, the degree of binding of an anti-DENV E protein antibody to an unrelated protein that is not a DENVE protein is less than about 10% of the binding of the antibody to the DENVE protein, as measured, for example, by radioimmunoassay (RIA). Is. In certain embodiments, the antibody that binds to the DENV and / or DENV E protein has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (eg, , 10 ^-8 M or less, for example 10 ^-8 M to 10 ^{-13 M, 10 -9 M to 10 -13} M). In certain embodiments, the anti-DENV antibody binds to a DENV epitope conserved between DENVs from different serotypes. In certain embodiments, the anti-DENVE protein antibody binds to an epitope of the DENVE protein that is conserved among the DENVE proteins from different serotypes.

The terms "anti-Zika virus antibody," "anti-Zika antibody," "anti-ZIKV antibody," and "antibody that binds to Zika virus" refer to antibodies that can bind to Zika virus with sufficient affinity, and as a result. , The antibody is useful as a diagnostic, prophylactic and / or therapeutic agent when targeting Zika virus. The terms "anti-ZIKV antibody" and "anti-DENV antibody" can be used interchangeably to refer to antibodies that can bind to both ZIKV virus and DENV.

The term "antibody" is used in the broadest sense herein, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments as long as they exhibit the desired antigen-binding activity. Includes various antibody structures including, but not limited to, these.

"Antibody-dependent cellular cytotoxicity" or "ADCC" is a secretion bound to an Fc receptor (FcR) present on certain cytotoxic cells (eg, NK cells, neutrophils, and macrophages). Ig refers to a form of cytotoxicity that causes these cytotoxic effector cells to specifically bind to antigen-carrying target cells and subsequently kill the target cells with cellular toxins. Primary cell NK cells for mediating ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, as described in US Pat. No. 5,500,362, 5,821,337, or US Pat. No. 6,737,056 (Presta). Effector cells useful for such assays include PBMC cells and NK cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, eg, in an animal model as disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998). ..

"Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of the intact antibody capable of binding to the antigen to which the intact antibody binds. Examples of antibody fragments are, but are not limited to, Fv, Fab, Fab', Fab'-SH, F (ab') ₂ ; diabody; linear antibody; single chain antibody molecule (eg scFv); And multispecific antibodies formed from antibody fragments.

"Antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of a reference antibody to its antigen in a competitive assay, and conversely, a reference antibody blocks the binding of an antibody to its antigen in a competitive assay. do. An exemplary competitive assay is provided herein.

"C1q" is a polypeptide containing a binding site for the Fc region of an immunoglobulin. C1q, together with two serine proteases C1r and C1s, forms complex C1 which is the first component of the complement-dependent cytotoxicity (CDC) pathway. Human C1q can be purchased commercially, for example, from Quidel (San Diego, CA).

The term "chimeric" antibody refers to an antibody in which part of a heavy chain and / or light chain is derived from a particular source or species and the rest of the heavy chain and / or light chain is derived from a different source or species.

An antibody "class" refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which are subclasses (isotypes) such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , And can be further classified into _{IgA 2.} The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

"Complement-dependent cytotoxicity" or "CDC" refers to lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that is bound to the cognate antigen. To assess complement activation, a CDC assay can be performed, eg, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Polypeptide variants with modified Fc region amino acid sequences (polypeptides with mutant Fc regions) and increased or decreased C1q binding capacity are described, for example, in US Pat. No. 6,194,551 B1 and WO 1999/51642. NS. See also, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or impedes cell function and / or causes cell death or destruction. Cytotoxic agents are, but are not limited to, radioisotopes (eg ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P. , ²¹² Pb, and Lu radioisotopes); chemotherapeutic agents or chemotherapeutic agents (eg methotrexate, adriamycin, binca alkaloids (vincristine, vinblastin, etoposide), doxorubicin, melfaran, mitomycin C, chlorambusyl, daunorubicin, or Other intercalating agents); growth inhibitors; enzymes such as nucleic acid degrading enzymes and fragments thereof; antibiotics; eg, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (fragments thereof) Toxins (including and / or variants); and various anti-tumor or anti-cancer agents disclosed below.

"Effector function" refers to the biological activity resulting from the Fc region of an antibody, which varies by antibody isotype. Examples of antibody effector function include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell injury. -mediated cytotoxicity: ADCC); phagocytosis; down-regulation of cell surface receptors (eg, B cell receptors); and B cell activation.

An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount at a required dose and over a required period of time that is effective in achieving the desired therapeutic or prophylactic result.

The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen and comprises certain amino acids that come into direct contact with the antibody. The epitope determinant can include chemically active surface molecules such as amino acids, sugar side chains, phosphoryl groups or sulfonyl groups and have specific three-dimensional structural and / or specific charge properties. Can be done. In general, an antibody specific for a particular target antigen preferentially recognizes an epitope on that target antigen in a complex mixture of proteins and / or macromolecules.

"Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds to an IgG antibody (gamma receptor) and forms the receptors of the FcγRI, FcγRII, and FcγRIII subclasses by allelic variants and alternative splicing of these receptors. Including, including. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domain. The activation receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15: 203-234 (1997).) FcR is, for example, Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et. Al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126: 330-41 (1995). Other FcRs, including those identified in the future, are also included in the term "FcR" herein.

The term "Fc receptor" or "FcR" also refers to the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249. (1994)) and includes the neonatal receptor FcRn, which is responsible for the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (eg, Ghetie and Ward., Immunol. Today 18 (12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15 (7): 637- 640 (1997); Hinton et al., J. Biol. Chem. 279 (8): 6213-6216 (2004); see WO2004 / 92219 (Hinton et al.)). In vivo binding to human FcRn and serum half-life of human FcRn high affinity binding polypeptides are administered, for example, in transgenic mice expressing human FcRn or in transfected human cell lines or by polypeptides with a mutant Fc region. Can be measured in primates. WO2000 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See also Shields et al. J. Biol. Chem. 9 (2): 6591-6604 (2001), for example.

As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes the Fc region of a native sequence and the mutant Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446 to 447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc or constant region is Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Follow the EU numbering system (also known as the EU index) described in MD 1991.

The term "Fc region-containing antibody" refers to an antibody containing an Fc region. The C-terminal lysine (residues 447) or glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant manipulation of the nucleic acid encoding the antibody. Therefore, a composition comprising an antibody having an Fc region according to the present invention may be an antibody with G446-K447, an antibody with G446 without K447, an antibody with G446-K447 completely removed, or three types of antibodies. May include a mixture.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. A variable domain FR usually consists of four FR domains: FR1, FR2, FR3, and FR4. Correspondingly, the sequences of HVR and FR usually appear in VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.

The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and have a structure substantially similar to that of a native antibody, or are defined herein. An antibody having a heavy chain containing an Fc region.

The "functional Fc region" includes the "effector function" of the natural sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (eg, B cell receptors: BCR) and the like. Such effector function generally requires the Fc region to be combined with a binding domain (eg, an antibody variable domain) and is evaluated using, for example, the various assays disclosed herein. obtain.

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which foreign nucleic acids have been introduced, including progeny of such cells. .. Host cells include "transformants" and "transformants", which include primary transformants and progeny derived from those cells regardless of the number of passages. The offspring do not have to be exactly the same in the content of the parent cell and nucleic acid and may contain mutations. Variant progeny with the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or an antibody derived from a non-human source using a human antibody repertoire or other human antibody coding sequence. This definition of human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

The "Human Consensus Framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Usually, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Usually, the sequence subgroups are those in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one aspect, for VL, the subgroup is the subgroup κI by Kabat et al. Above. In one aspect, for VH, the subgroup is Subgroup III by Kabat et al. Above.

A "humanized" antibody is a chimeric antibody that comprises an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In some embodiments, the humanized antibody comprises substantially all of at least one, typically two variable domains, in which all or substantially all HVRs (eg, CDRs) are non-existent. Corresponds to those of human antibodies, and all or substantially all FRs correspond to those of human antibodies. The humanized antibody may optionally include at least a portion of the antibody constant region derived from the human antibody. The "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.

As used herein, the terms "hypervariable region" or "HVR" are hypervariable in sequence ("complementarity determining regions" or "CDR") and / or structurally defined loops ("hypervariable"). Refers to each region of the variable domain of an antibody that forms a loop ") and / or contains an antigen contact residue ("antigen contact"). Usually, the antibody contains 6 HVRs: 3 for VH (H1, H2, H3) and 3 for VL (L1, L2, L3). Illustrative HVRs herein include:
(a) At amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) The resulting hypervariable loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));
(b) At amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3). The resulting CDR (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) At amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3). Antigen contact that occurs (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996));
(d) HVR Amino Acid Residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49 A combination of (a), (b), and / or (c), including -65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., Supra.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, the heterologous molecule including, but not limited to, a cytotoxic agent.

The "individual" or "subject" is a mammal. Mammals are, but are not limited to, domestic animals (eg, cows, sheep, cats, dogs, horses), primates (eg, non-human primates such as humans and monkeys), rabbits, and , Includes primates (eg, mice and rats). In certain embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been isolated from its original environmental components. In some embodiments, the antibody is subjected to, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatograph (eg, ion exchange or reverse phase HPLC). Measured and purified to a purity greater than 95% or 99%. See, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007) for a review of methods for assessing antibody purity.

An "isolated" nucleic acid is a nucleic acid molecule that has been isolated from its original environmental components. An isolated nucleic acid contains a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is on a chromosome that is extrachromosomal or different from its original chromosomal location. Exists in position.

"An isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, on one vector or separate vectors. Includes nucleic acid molecules that are present and nucleic acid molecules that are present at one or more positions in the host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up the population are possible mutant antibodies (eg, mutant antibodies that contain naturally occurring mutations, or mutant antibodies that occur during the production of monoclonal antibody preparations, such variants are usually slightly present. Except for the amount present), it binds to the same and / or the same epitope. Each monoclonal antibody in a monoclonal antibody preparation is for a single determinant on an antigen, as opposed to a polyclonal antibody preparation that typically comprises different antibodies against different determinants (epitopes). Therefore, the modifier "monoclonal" should not be construed as requiring the production of an antibody by any particular method, indicating the characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies. For example, the monoclonal antibody used according to the present invention is not limited to these, but is a hybridoma method, a recombinant DNA method, a phage display method, and a transgenic animal containing all or a part of the human immunoglobulin locus. It may be made by a variety of methods, including methods that utilize, such methods and other exemplary methods for making monoclonal antibodies are described herein.

"Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. The naked antibody may be present in the pharmaceutical formulation.

"Natural antibody" refers to an immunoglobulin molecule with a variety of naturally occurring structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical disulfide-bonded light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, followed by a stationary light chain (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The "natural sequence Fc region" includes an amino acid sequence that is the same as the amino acid sequence of the Fc region found in nature. The natural sequence human Fc region is the natural sequence human IgG1 Fc region (non-A and A allotype); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, and the natural Includes those variants present in.

The term "package insert" is commonly included in commercial packaging of therapeutic products and contains information about indications, dosages, doses, dosage regimens, concomitant therapies, contraindications, and / or warnings regarding the use of such therapeutic products. It is used to refer to the instruction manual.

"Percent (%) amino acid sequence identity" to a reference polypeptide sequence is sequenced to obtain maximum percent sequence identity and, if necessary, after introduction of a gap and any conservative substitution. It is defined as the percentage ratio of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence when not considered part of the identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed by various methods within the skill of the art, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx). This can be achieved by using publicly available computer software such as Co., Ltd.). One of skill in the art can determine the appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment over the overall length of the sequence being compared.

The ALIGN-2 Sequence Comparison Computer Program is the work of Genetec, the source code of which was submitted to the United States Copyright Office (Washington, DC, 20559) along with user documentation under the United States Copyright Registration Number TXU510087. It is registered. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California and may be compiled from source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not fluctuate. In situations where ALIGN-2 is used for amino acid sequence comparison,% amino acid sequence identity (or given amino acid sequence) of a given amino acid sequence A to, to, or to a given amino acid sequence B. A given amino acid sequence A, which has or contains some% amino acid sequence identity to, to, or to B) is calculated as follows:
Part 100 times X / Y where X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 as identical matches in the A and B alignments of that program, where Y is in B. Is the total number of amino acid residues in. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of A to B is not equal to the% amino acid sequence identity of B to A. Let's go. Unless otherwise stated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

The term "pharmaceutical preparation" is a preparation in a form in which the biological activity of the active ingredient contained therein can exert its effect, and to the extent unacceptable to the subject to which the preparation is administered. A preparation that does not contain additional toxic elements.

"Pharmaceutically acceptable carrier" refers to an ingredient other than the active ingredient in a pharmaceutical formulation that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, the term "DENV E protein" is a natural term from any DENV serotype, including DENV-1, DENV-2, DENV-3, and DENV-4, unless otherwise indicated. Refers to the DENV E protein. The DENV genome consists of three structural proteins (capsid (C), premembrane / membrane (prM / M), and envelope (E)) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and Code NS5). The E protein is a glycoprotein of about 55 kDa and exists as a heterodimer with the PrM protein before the maturation of virions. X-ray crystallographic studies of the extracellular domain of the E protein revealed three different β-barrel domains linked to the viral membrane by a spiral stem anchor and two antiparallel transmembrane domains. Domain III (EDIII) has an immunoglobulin-like fold, which has been suggested to play an important role in receptor interaction. Domain II (EDII) is an elongated domain consisting of two long finger-like structures, including a highly conserved 13-amino acid fusion loop (EDII-FL) at the tip, which is a membrane fusion and dimer of E protein. Involved in the transformation. The central domain (domain I; EDI) is a 9-stranded β-barrel linked to EDID and EDII by one and four mobile linkers, respectively. E-proteins are important for viral assembly, receptor binding, invasion, viral fusion, and possibly immune escape in the viral life cycle, and are therefore required to take several different conformations and arrangements on viral particles. It is a dynamic protein that is said to be. The term includes not only the unprocessed "full length" DENVE protein, but also any form of DENVE protein resulting from intracellular processing. The term also includes naturally occurring variants of the DENVE protein, such as serotype variants or quasispecies.

As used herein, "ZIKV" is a member of the Flaviviridae virus. It is transmitted by Aedes mosquitoes that are active during the day, such as Aedes aegypti and Aedes albopictus. The ZIKV genome encodes a single polyprotein of 3419 amino acids, which are functional domains by viral serine proteases and cellular furin proteases, namely capsids (C), precursor membranes (prM). ), Envelope (E) and 7 nonstructural proteins (NS).

As used herein, the terms "substantially reduced," "substantially increased," or "substantially different" are two numbers (generally, a number associated with a molecule and a number associated with a molecule. Refers to a sufficiently high degree of difference between (other numbers associated with the reference / comparative molecule), so that one of ordinary skill in the art will measure the difference between these two values by the value (eg, Kd value). It would be considered statistically significant in the context of scientific features.

As used herein, "treatment" (and its grammatical derivatives such as "treat", "treat", etc.) is clinically intended to alter the natural course of the individual being treated. It means intervention and can be performed both for prevention and during the course of the clinical condition. The desired effects of treatment are, but are not limited to, prevention of the onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological effects of the disease, prevention of metastasis, prevention of the disease, of the disease. Includes reduced rate of progression, recovery or alleviation of disease state, and ameliorated or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of a disease or slow the progression of a disease.

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody involved in binding the antibody to an antigen. The heavy and light chain variable domains of native antibodies (VH and VL, respectively) are similar, with each domain usually containing four conserved framework regions (FR) and three hypervariable regions (HVR). Has a structure. (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) One VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains using the VH or VL domains from antibodies that bind to that antigen, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

A "mutated Fc region" includes an amino acid sequence that differs from that of the native sequence Fc region by at least one amino acid modification (modification), preferably one or more amino acid substitutions. Preferably, the mutant Fc region has at least one amino acid substitution in the native sequence Fc region or in the Fc region of the parent polypeptide, eg, about 1 to, as compared to the native sequence Fc region or the Fc region of the parent polypeptide. It has about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions. The mutant Fc regions herein are preferably at least about 80% homologous to the native sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology to them, more preferably. Have at least about 95% homology with them.

As used herein, the term "vector" refers to a nucleic acid molecule to which it can augment another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. Certain vectors can result in the expression of nucleic acids to which they are operably linked. Such vectors are also referred to herein as "expression vectors."

II. Compositions and Methods In one aspect, the invention is based in part on anti-DENV antibodies and their use. In certain embodiments, antibodies that bind to DENV and / or DENV E proteins are provided. The antibodies of the present invention are useful, for example, in the diagnosis or treatment of DENV infection.

In one aspect, the invention is based in part on a polypeptide containing a mutant Fc region and its use. In certain embodiments, a polypeptide comprising a mutant Fc region with substantially reduced FcγR binding activity is provided. In certain embodiments, a polypeptide comprising a mutant Fc region that does not have substantially reduced C1q binding activity is provided. In certain embodiments, the polypeptides of the invention are antibodies. Polypeptides containing the mutant Fc region of the present invention are useful, for example, in the diagnosis or treatment of viral infections.

In one aspect, the invention is based in part on the anti-ZIKV antibody and its use. In certain embodiments, an antibody that binds to ZIKV is provided. In certain embodiments, an anti-ZIKV antibody that is cross-reactive with DENV is provided.

In one aspect, the invention is based in part on a method of treating or preventing Zika infection, which comprises the step of administering an antibody that binds to ZIKV. In one aspect, the invention is based in part on a method of blocking the transmission of Zika virus from a pregnant mother to the foetation, including the step of administering an antibody that binds to ZIKV. In one aspect, the invention is based in part on a method of preventing congenital Zika syndrome, which comprises the step of administering an antibody that binds to ZIKV. In one aspect, the invention is based in part on a method of suppressing the loss of fetal weight (eg, whole body, head, etc.), including the step of administering an antibody that binds to ZIKV.

In one example, the term "congenital Zika syndrome" refers to a distinct pattern of birth defects typical of prenatally infected fetuses and newborns. As will be appreciated by those skilled in the art, subjects with congenital deca syndrome may exhibit symptoms such as, but not limited to: congenital dysgenesis, microcephaly, partial skull. Severe microcephaly that collapses, loss of brain tissue with certain patterns of brain damage such as subcortical calcification, damage to the fundus such as speckled scars and localized pigmented retinal spots, varus foot Or congenital contractions such as joint contractions, hypertonia that limits physical activity immediately after birth, etc. Administration of the antibodies described herein is believed to prevent at least one (or at least two, or at least three, or at least four, or all) symptoms of congenital deca syndrome. For example, the antibodies described herein prevent fetal or neonatal infection of prenatal Zika virus, thereby suppressing fetal weight loss (eg, systemic, head, etc.).

In one aspect, the term "anti-DENV antibody" described in this section (II. Compositions and Methods) is an antibody capable of binding ZIKV ("anti-DENV antibody" as long as the antibody can bind to both DENV and ZIKV. Can be used to refer to "ZIKV antibody"). In one aspect, the invention provides a method of treating or preventing Zika infection, comprising administering an antibody that binds to Zika virus (“anti-ZIKV antibody”). In one aspect, the invention provides a method of blocking the transmission of Zika virus from a pregnant mother to a foetation, comprising administering an antibody that binds to Zika virus.

A. An exemplary anti-DENV antibody and polypeptide containing a variant Fc region In one aspect, the invention provides an isolated antibody that binds to DENV and / or DENVE protein. In certain embodiments, the anti-DENV antibody blocks the binding of the DENV and / or DENVE protein to the host cell. In certain embodiments, the anti-DENV antibody inhibits DENV invasion into the host cell. In certain embodiments, the anti-DENV antibody binds to the entire DENV particle. In a further aspect, the antibody binds to the entire DENV particle rather than to the monomeric DENVE protein. In certain embodiments, the anti-DENV antibodies of the invention are DENV serotype 1 (DENV-1), DENV serotype 2 (DENV-2), DENV serotype 3 (DENV-3), and DENV serotype 4 (DENV). -4) Binds and / or neutralizes at least one, at least two, at least three, or all four DENV serum types selected from the group consisting of. In certain embodiments, the anti-DENV antibody is at least one, at least two, at least three, or all four selected from the group consisting of DENV-1, DENV-2, DENV-3, and DENV-4. It binds to and / or neutralizes the DENV E protein from the DENV serotype. "Serotype" refers to a clearly distinguishable variation within a viral species.

In one aspect, the invention provides an isolated antibody that binds to ZIKV. In certain embodiments, the anti-ZIKV antibody blocks ZIKV from binding to the host cell. In certain embodiments, the anti-ZIKV antibody inhibits ZIKV from invading the host cell. In certain embodiments, the anti-ZIKV antibody is cross-reactive with DENV. In one aspect, the invention comprises HVR-H1 comprising any one amino acid sequence of (a) SEQ ID NO: 11-12; (b) comprising any one amino acid sequence of SEQ ID NO: 13-15. HVR-H2; (c) HVR-H3 containing any one amino acid sequence of SEQ ID NO: 16-20; (d) HVR-L1 containing any one amino acid sequence of SEQ ID NO: 21-23; Selected from (e) HVR-L2 containing any one amino acid sequence of SEQ ID NO: 24-26; and (f) HVR-L3 containing any one amino acid sequence of SEQ ID NO: 27-30. Provide anti-DENV antibodies comprising at least one, two, three, four, five, or six HVRs.

In another aspect, the invention comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) SEQ ID NO: HVR-H3 containing 42 amino acid sequences; (d) SEQ ID NO: HVR-L1 containing 43 amino acid sequences; (e) SEQ ID NO: HVR-L2 containing 44 amino acid sequences; and (f) SEQ ID Provided is an anti-DENV antibody comprising at least one, two, three, four, five, or six HVRs selected from HVR-L3 containing the amino acid sequence NO: 45.

In another aspect, the invention comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) SEQ ID NO: HVR-H3 containing 20 amino acid sequences; (d) SEQ ID NO: HVR-L1 containing 23 amino acid sequences; (e) SEQ ID NO: HVR-L2 containing 26 amino acid sequences; and (f) SEQ ID NO: Provides anti-DENV antibodies comprising at least one, two, three, four, five, or six HVRs selected from HVR-L3 containing an amino acid sequence of 30.

In another aspect, the invention comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) SEQ ID NO: HVR-H3 containing 20 amino acid sequences; (d) SEQ ID NO: HVR-L1 containing 21 amino acid sequences; (e) SEQ ID NO: HVR-L2 containing 24 amino acid sequences; and (f) SEQ ID NO: Provides anti-DENV antibodies comprising at least one, two, three, four, five, or six HVRs selected from HVR-L3 containing the amino acid sequence of 27.

In one aspect, the invention comprises HVR-H1 comprising any one amino acid sequence of (a) SEQ ID NO: 11-12; (b) comprising any one amino acid sequence of SEQ ID NO: 13-15. HVR-H2; and (c) SEQ ID NO: An antibody containing at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing any one amino acid sequence of 16-20. I will provide a. In one aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NO: 16-20. In another aspect, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NO: 16-20 and HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 27-30. .. In a further aspect, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NO: 16-20, HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 27-30, and SEQ. ID NO: Contains HVR-H2 containing the amino acid sequence of any one of 13-15. In a further aspect, the antibody comprises HVR-H1 comprising any one amino acid sequence of (a) SEQ ID NO: 11-12; (b) comprising any one amino acid sequence of SEQ ID NO: 13-15. HVR-H2; and (c) SEQ ID NO: Includes HVR-H3 containing the amino acid sequence of any one of 16-20.

In another aspect, the invention comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; and (c) SEQ ID NO. : Provide an antibody containing at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing 42 amino acid sequences. In one aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42. In another aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41. including. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; and (c) SEQ ID NO: Includes HVR-H3, which contains 42 amino acid sequences.

In another aspect, the invention comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; and (c) SEQ ID NO. : Provide an antibody containing at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing 20 amino acid sequences. In one aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20. In another aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In another aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. In a further aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15. including. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15. including. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; and (c) SEQ ID NO: Includes HVR-H3, which contains 20 amino acid sequences.

In another aspect, the present invention comprises HVR-L1 comprising any one amino acid sequence of (a) SEQ ID NO: 21-23; (b) any one amino acid sequence of SEQ ID NO: 24-26. Includes HVR-L2; and (c) SEQ ID NO: Contains at least one, at least two, or all three VL HVR sequences selected from HVR-L3 containing any one amino acid sequence of 27-30. Provide an antibody. In one aspect, the antibody comprises HVR-L1 comprising any one amino acid sequence of (a) SEQ ID NO: 21-23; (b) comprising any one amino acid sequence of SEQ ID NO: 24-26. HVR-L2; and (c) Contains HVR-L3 containing the amino acid sequence of any one of SEQ ID NO: 27-30.

In another aspect, the invention comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) SEQ ID NO. : Provide an antibody containing at least one, at least two, or all three VL HVR sequences selected from HVR-L3 containing 45 amino acid sequences. In one aspect, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) SEQ ID NO: Includes HVR-L3 containing 45 amino acid sequences.

In another aspect, the invention comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) SEQ ID NO. : Provide an antibody containing at least one, at least two, or all three VL HVR sequences selected from HVR-L3 containing 30 amino acid sequences. In one aspect, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) SEQ ID NO: Includes HVR-L3 containing 30 amino acid sequences.

In another aspect, the antibody of the present invention is any of (a) (i) HVR-H1 containing an amino acid sequence of any one of SEQ ID NOs: 11-12, and (ii) SEQ ID NO: 13-15. At least one, at least two, or all three selected from HVR-H2 containing one amino acid sequence and HVR-H3 containing any one amino acid sequence of (iii) SEQ ID NO: 16-20. A VH domain containing a VH HVR sequence and one of (b) (i) HVR-L1 containing an amino acid sequence of any one of SEQ ID NOs: 21 to 23 and (ii) SEQ ID NO: 24 to 26. At least one, at least two, or all three VL HVRs selected from HVR-L2 containing an amino acid sequence and HVR-L3 containing any one amino acid sequence of (iii) SEQ ID NO: 27-30. Includes the VL domain containing the sequence.

In another aspect, the antibodies of the invention are (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 40, (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 41, and ( iii) SEQ ID NO: A VH domain containing at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing the amino acid sequence of 42, and (b) (i) SEQ ID NO. At least selected from HVR-L1 containing the amino acid sequence of: 43, HVR-L2 containing the amino acid sequence of (ii) SEQ ID NO: 44, and HVR-L3 containing the amino acid sequence of (iii) SEQ ID NO: 45. Includes a VL domain containing one, at least two, or all three VL HVR sequences.

In another aspect, the antibodies of the invention are (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 12, (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 15, and ( iii) SEQ ID NO: A VH domain containing at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing an amino acid sequence of 20 and (b) (i) SEQ ID NO. At least selected from HVR-L1 containing the amino acid sequence of: 23, HVR-L2 containing the amino acid sequence of (ii) SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of (iii) SEQ ID NO: 30. Includes a VL domain containing one, at least two, or all three VL HVR sequences.

In another aspect, the antibodies of the invention are (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 12, (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 15, and ( iii) SEQ ID NO: A VH domain containing at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing an amino acid sequence of 20 and (b) (i) SEQ ID NO. At least selected from HVR-L1 containing the amino acid sequence of: 21, HVR-L2 containing the amino acid sequence of (ii) SEQ ID NO: 24, and HVR-L3 containing the amino acid sequence of (iii) SEQ ID NO: 27. Includes a VL domain containing one, at least two, or all three VL HVR sequences.

In another aspect, the invention comprises HVR-H1 comprising any one amino acid sequence of (a) SEQ ID NO: 11-12; (b) any one amino acid sequence of SEQ ID NO: 13-15. HVR-H2 containing; (c) HVR-H3 containing any one amino acid sequence of SEQ ID NO: 16-20; (d) HVR-L1 containing any one amino acid sequence of SEQ ID NO: 21-23 (E) HVR-L2 containing any one amino acid sequence of SEQ ID NO: 24-26; and (f) antibody containing HVR-L3 containing any one amino acid sequence of SEQ ID NO: 27-30 I will provide a.

In another aspect, the invention comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) SEQ ID NO: HVR-H3 containing 42 amino acid sequences; (d) SEQ ID NO: HVR-L1 containing 43 amino acid sequences; (e) SEQ ID NO: HVR-L2 containing 44 amino acid sequences; and (f) SEQ ID Provided is an antibody containing HVR-L3 containing the amino acid sequence of NO: 45.

In another aspect, the invention comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) SEQ ID NO: HVR-H3 containing 20 amino acid sequences; (d) SEQ ID NO: HVR-L1 containing 23 amino acid sequences; (e) SEQ ID NO: HVR-L2 containing 26 amino acid sequences; and (f) SEQ ID Provided is an antibody containing HVR-L3 containing the amino acid sequence of NO: 30.

In another aspect, the invention comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) SEQ ID NO: HVR-H3 containing 20 amino acid sequences; (d) SEQ ID NO: HVR-L1 containing 21 amino acid sequences; (e) SEQ ID NO: HVR-L2 containing 24 amino acid sequences; and (f) SEQ ID Provided is an antibody containing HVR-L3 containing the amino acid sequence of NO: 27.

In certain embodiments, any one or more amino acids of the anti-DENV antibody described above are replaced at the following HVR positions: (a) at positions 2 and 4 in HVR-H1 (SEQ ID NO: 11). (B) 9th and 10th place in HVR-H2 (SEQ ID NO: 13); (c) 3rd, 14th and 16th place in HVR-H3 (SEQ ID NO: 16); (d) HVR -L1 (SEQ ID NO: 21) 5th and 8th; (e) HVR-L2 (SEQ ID NO: 24) 4th and 7th; and (f) HVR-L3 (SEQ ID NO: 24) In 27), 4th and 5th place.

In certain embodiments, the amino acid substitution of one or more anti-DENV antibodies is a conservative substitution, as provided herein. In certain embodiments, any one or more of the following substitutions can be made in any combination: (a) HVR-H1 (SEQ ID NO: 11), N2Y; I4M; (b) HVR-H2 (SEQ ID NO: 13) for T9R; A10R; (c) HVR-H3 (SEQ ID NO: 16) for R3E; R14F; G16D or L; (d) HVR-L1 (SEQ ID NO: 21) , D5E; K8Q; (e) HVR-L2 (SEQ ID NO: 24), N4E; T7F; and (f) HVR-L3 (SEQ ID NO: 27), D4S or E; D5A.

All possible combinations of the above substitutions are SEQ ID NOs: 40, 41, 42, 43 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, respectively. , 44 and 45 consensus sequences.

In a further aspect, the anti-DENV antibody of the invention is HVR-H1, which comprises the amino acid sequence of (i) SEQ ID NO: 11, HVR-H2, which comprises the amino acid sequence of (ii) SEQ ID NO: 13, and (iii) SEQ. HVR-H3 containing the amino acid sequence of ID NO: 16, HVR-L1 containing the amino acid sequence of (iv) SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of (v) SEQ ID NO: 24, and (vi) ) SEQ ID NO: Not an antibody containing HVR-L3 containing the amino acid sequence of 27.

In another aspect, the invention is derived from (a) HVR-H1 from any one VH sequence of SEQ ID NO: 2-6; (b) derived from any one VH sequence of SEQ ID NO: 2-6. HVR-H2; (c) SEQ ID NO: HVR-H3 derived from any one VH sequence of 2 to 6; (d) SEQ ID NO: HVR-L1 derived from any one of VL sequences of 8 to 10. Selected from (e) HVR-L2 from any one VL sequence of SEQ ID NO: 8-10; and (f) HVR-L3 from any one VL sequence of SEQ ID NO: 8-10 Provide anti-DENV antibodies, including at least one, two, three, four, five, or six HVRs.

In another aspect, the invention is derived from (a) HVR-H1 from any one VH sequence of SEQ ID NO: 2-6; (b) derived from any one VH sequence of SEQ ID NO: 2-6. HVR-H2; (c) HVR-H3 from any one VH sequence of SEQ ID NO: 2-6; (d) HVR-L1 from any one VL sequence of SEQ ID NO: 7; e) HVR-L2 from any one VL sequence of SEQ ID NO: 7; and (f) at least one selected from HVR-L3 from any one VL sequence of SEQ ID NO: 7, 2 Provide anti-DENV antibodies containing one, three, four, five, or six HVRs.

In another aspect, the invention is: (a) HVR-H1 from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 from the VH sequence of SEQ ID NO: 6; (c) SEQ ID NO: HVR-H3 from VH sequence of 6; (d) HVR-L1 from VL sequence of 10; (e) SEQ ID NO: HVR-L2 from VL sequence of 10; and (f) SEQ ID Provide anti-DENV antibodies comprising at least one, two, three, four, five, or six HVRs selected from HVR-L3s from the NO: 10 VL sequence.

In another aspect, the invention is: (a) HVR-H1 from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 from the VH sequence of SEQ ID NO: 6; (c) SEQ ID NO: HVR-H3 from VH sequence of 6; (d) HVR-L1 from VL sequence of 7; (e) SEQ ID NO: HVR-L2 from VL sequence of 7; and (f) SEQ ID Provide anti-DENV antibodies comprising at least one, two, three, four, five, or six HVRs selected from HVR-L3s from the NO: 7 VL sequence.

In one aspect, the invention is derived from (a) HVR-H1 from any one VH sequence of SEQ ID NO: 2-6; (b) derived from any one VH sequence of SEQ ID NO: 2-6. HVR-H2; and (c) SEQ ID NO: Antibodies containing at least one, at least two, or all three VH HVR sequences selected from HVR-H3 from any one VH sequence of 2-6. I will provide a. In one aspect, the antibody comprises HVR-H3 from any one VH sequence of SEQ ID NO: 2-6. In another aspect, the antibody comprises HVR-H3 from any one VH sequence of SEQ ID NO: 2-6 and HVR-L3 from any one VL sequence of SEQ ID NO: 8-10. .. In another aspect, the antibody comprises HVR-H3 from any one VH sequence of SEQ ID NO: 2-6 and HVR-L3 from any one VL sequence of SEQ ID NO: 7. In a further embodiment, the antibody is HVR-H3 from any one VH sequence of SEQ ID NO: 2-6, HVR-L3 from any one VL sequence of SEQ ID NO: 8-10, and SEQ. ID NO: Contains HVR-H2 from any one of the VH sequences 2-6. In a further embodiment, the antibody is HVR-H3 from any one VH sequence of SEQ ID NO: 2-6, HVR-L3 from any one VL sequence of SEQ ID NO: 7, and SEQ ID NO. : Includes HVR-H2 from any one of the 2-6 VH sequences. In a further embodiment, the antibody is derived from (a) HVR-H1 from any one VH sequence of SEQ ID NO: 2-6; (b) from any one VH sequence of SEQ ID NO: 2-6. HVR-H2; and (c) SEQ ID NO: Includes HVR-H3 from any one of the VH sequences 2-6.

In one aspect, the invention is: (a) HVR-H1 from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 from the VH sequence of SEQ ID NO: 6; (c) SEQ ID NO: 6 Provided is an antibody comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3s derived from the VH sequences of. In one aspect, the antibody comprises HVR-H3 from the VH sequence of SEQ ID NO: 6. In another aspect, the antibody comprises HVR-H3 from the VH sequence of SEQ ID NO: 6 and HVR-L3 from the VL sequence of SEQ ID NO: 10. In another aspect, the antibody comprises HVR-H3 from the VH sequence of SEQ ID NO: 6 and HVR-L3 from the VL sequence of SEQ ID NO: 7. In a further embodiment, the antibody is HVR-H3 from the VH sequence of SEQ ID NO: 6, HVR-L3 from the VL sequence of SEQ ID NO: 10, and HVR-H2 from the VH sequence of SEQ ID NO: 6. including. In a further embodiment, the antibody is HVR-H3 from the VH sequence of SEQ ID NO: 6, HVR-L3 from the VL sequence of SEQ ID NO: 7, and HVR-H2 from the VH sequence of SEQ ID NO: 6. including. In a further embodiment, the antibody comprises (a) HVR-H1 from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 from the VH sequence of SEQ ID NO: 6; and (c) SEQ ID NO: Contains HVR-H3 from 6 VH sequences.

In another aspect, the invention is derived from (a) HVR-L1 from any one VL sequence of SEQ ID NO: 8-10; (b) derived from any one VL sequence of SEQ ID NO: 8-10. HVR-L2; and (c) SEQ ID NO: Includes at least one, at least two, or all three VL HVR sequences selected from HVR-L3s derived from any one of the VL sequences 8-10. Provide an antibody. In one aspect, the antibody is derived from HVR-L1 from any one VL sequence of (a) SEQ ID NO: 8-10; (b) derived from any one VL sequence of SEQ ID NO: 8-10. HVR-L2; and (c) SEQ ID NO: Includes HVR-L3 from any one of the VL sequences 8-10.

In another aspect, the invention is: (a) HVR-L1 from the VL sequence of SEQ ID NO: 10; (b) HVR-L2 from the VL sequence of SEQ ID NO: 10; and (c) SEQ ID NO. : Provide an antibody comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3s derived from 10 VL sequences. In one aspect, the antibody is (a) HVR-L1 from the VL sequence of SEQ ID NO: 10; (b) HVR-L2 from the VL sequence of SEQ ID NO: 10; and (c) SEQ ID NO: Contains HVR-L3 from 10 VL sequences.

In another aspect, the antibody of the present invention is HVR-H1 derived from any one of the VH sequences of (a) (i) SEQ ID NO: 2 to 6, and (ii) SEQ ID NO: 2 to 6. At least one, at least two, or all three selected from HVR-H2 from one VH sequence and HVR-H3 from any one VH sequence of (iii) SEQ ID NO: 2-6. VH domain containing VH HVR sequence and HVR-L1 derived from (b) (i) SEQ ID NO: 8 to any one of VL sequences, (ii) SEQ ID NO: any one of 8 to 10. HVR-L2 from VL sequence and (iii) SEQ ID NO: At least one, at least two, or all three VL HVRs selected from HVR-L3 from any one of the VL sequences of 8-10. Includes the VL domain containing the sequence.

In another aspect, the antibody of the present invention is HVR-H1 derived from any one of the VH sequences of (a) (i) SEQ ID NO: 2 to 6, and (ii) SEQ ID NO: 2 to 6. At least one, at least two, or all three selected from HVR-H2 from one VH sequence and HVR-H3 from any one VH sequence of (iii) SEQ ID NO: 2-6. The VH domain containing the VH HVR sequence and HVR-L1 derived from any one of the VL sequences of (b) (i) SEQ ID NO: 7 and (ii) derived from the VL sequence of any one of SEQ ID NO: 7. With a VL domain containing at least one, at least two, or all three VL HVR sequences selected from HVR-L2 and HVR-L3 from any one VL sequence of (iii) SEQ ID NO: 7. including.

In another aspect, the antibodies of the invention are (a) (i) HVR-H1 from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 from the VH sequence of SEQ ID NO: 6, and ( iii) SEQ ID NO: A VH domain containing at least one, at least two, or all three VH HVR sequences selected from HVR-H3s derived from the 6 VH sequences, and (b) (i) SEQ ID NO. At least selected from: HVR-L1 from 10 VL sequence, (ii) HVR-L2 from SEQ ID NO: 10 VL sequence, and (iii) HVR-L3 from SEQ ID NO: 10 VL sequence Includes a VL domain containing one, at least two, or all three VL HVR sequences.

In another aspect, the antibodies of the invention are (a) (i) HVR-H1 from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 from the VH sequence of SEQ ID NO: 6, and ( iii) SEQ ID NO: A VH domain containing at least one, at least two, or all three VH HVR sequences selected from HVR-H3s derived from the 6 VH sequences, and (b) (i) SEQ ID NO. At least selected from: HVR-L1 from the VL sequence of 7, (ii) HVR-L2 from the VL sequence of SEQ ID NO: 7, and (iii) HVR-L3 from the VL sequence of SEQ ID NO: 7. Includes a VL domain containing one, at least two, or all three VL HVR sequences.

In another aspect, the invention is derived from (a) HVR-H1 from any one VH sequence of SEQ ID NO: 2-6; (b) derived from any one VH sequence of SEQ ID NO: 2-6. HVR-H2; (c) SEQ ID NO: HVR-H3 derived from any one VH sequence of 2 to 6; (d) SEQ ID NO: HVR-L1 derived from any one of VL sequences of 8 to 10. (E) SEQ ID NO: HVR-L2 from any one VL sequence of 8-10; and (f) SEQ ID NO: HVR-L3 from any one VL sequence of 8-10 I will provide a.

In another aspect, the invention is derived from (a) HVR-H1 from any one VH sequence of SEQ ID NO: 2-6; (b) derived from any one VH sequence of SEQ ID NO: 2-6. HVR-H2; (c) HVR-H3 from any one VH sequence of SEQ ID NO: 2-6; (d) HVR-L1 from any one VL sequence of SEQ ID NO: 7; Provided are antibodies comprising HVR-L2 from any one VL sequence of SEQ ID NO: 7; and (f) HVR-L3 from any one VL sequence of SEQ ID NO: 7.

In another aspect, the invention is: (a) HVR-H1 from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 from the VH sequence of SEQ ID NO: 6; (c) SEQ ID NO: HVR-H3 from VH sequence of 6; (d) HVR-L1 from VL sequence of 10; (e) SEQ ID NO: HVR-L2 from VL sequence of 10; and (f) SEQ ID An antibody containing HVR-L3 derived from the NO: 10 VL sequence is provided.

In another aspect, the invention is: (a) HVR-H1 from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 from the VH sequence of SEQ ID NO: 6; (c) SEQ ID NO: HVR-H3 from VH sequence of 6; (d) HVR-L1 from VL sequence of 7; (e) SEQ ID NO: HVR-L2 from VL sequence of 7; and (f) SEQ ID An antibody containing HVR-L3 derived from the VL sequence of NO: 7 is provided.

In a further aspect, the anti-DENV antibody of the invention is (i) HVR-H1 from the VH sequence of SEQ ID NO: 1; (ii) HVR-H2 from the VH sequence of SEQ ID NO: 1; (iii) SEQ. HVR-H3 from VH sequence with ID NO: 1; (iv) HVR-L1 from VL sequence with SEQ ID NO: 7; (v) HVR-L2 from VL sequence with SEQ ID NO: 7; and (vi) ) SEQ ID NO: 7 is not an antibody containing HVR-L3 derived from the VL sequence.

In any of the above embodiments, the anti-DENV antibody can be humanized. In one aspect, the anti-DENV antibody comprises HVR in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another aspect, the anti-DENV antibody comprises HVR in any of the above embodiments, and further comprises VH or VL comprising an FR sequence. In a further aspect, the anti-DENV antibody comprises the following heavy and / or light chain variable domain FR sequences. For heavy chain variable domains, FR1 contains the amino acid sequence of SEQ ID NO: 31, FR2 contains the amino acid sequence of SEQ ID NO: 32, and FR3 contains the amino acid sequence of SEQ ID NO: 33 or 34. FR4 contains the amino acid sequence of SEQ ID NO: 35. In the case of the light chain variable domain, FR1 contains the amino acid sequence of SEQ ID NO: 36, FR2 contains the amino acid sequence of SEQ ID NO: 37, FR3 contains the amino acid sequence of SEQ ID NO: 38, and FR4 contains the amino acid sequence of SEQ ID NO: 38. Contains the amino acid sequence of SEQ ID NO: 39.

In another aspect, the anti-DENV antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 for any one amino acid sequence of SEQ ID NO: 2-6. Includes heavy chain variable domain (VH) sequences with%, 98%, 99%, or 100% sequence identity. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to the reference sequence. An anti-DENV antibody containing a substitution (eg, conservative substitution), insertion, or deletion, but containing that sequence retains the ability to bind DENV. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in any one of SEQ ID NOs: 2-6. In certain embodiments, substitutions, insertions or deletions occur in the outer region of the HVR (ie, FR). Optionally, the anti-DENV antibody comprises a VH sequence of any one of SEQ ID NO: 2-6, including post-translational modifications. In certain embodiments, the VH comprises the amino acid sequence of any one of (a) SEQ ID NO: 11-12, HVR-H1, and (b) SEQ ID NO: 13-15. Contains one, two, or three HVRs selected from HVR-H2 containing, and (c) SEQ ID NO: HVR-H3 containing any one amino acid sequence of 16-20. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In another aspect, the anti-DENV antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 with respect to the amino acid sequence of SEQ ID NO: 6. Includes heavy chain variable domain (VH) sequences with% or 100% sequence identity. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to the reference sequence. An anti-DENV antibody containing a substitution (eg, conservative substitution), insertion, or deletion, but containing that sequence retains the ability to bind DENV. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and / or deleted at SEQ ID NO: 6. In certain embodiments, substitutions, insertions or deletions occur in the outer region of the HVR (ie, FR). Optionally, the anti-DENV antibody comprises a VH sequence of SEQ ID NO: 6, including post-translational modifications. In certain embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and (c) SEQ ID NO. : Contains one, two, or three HVRs selected from HVR-H3s containing 20 amino acid sequences. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In another aspect, the anti-DENV antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 for any one amino acid sequence of SEQ ID NO: 8-10. Includes light chain variable domain (VL) sequences with%, 98%, 99%, or 100% sequence identity. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to the reference sequence. An anti-DENV antibody containing a substitution (eg, conservative substitution), insertion, or deletion, but containing that sequence retains the ability to bind DENV. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted at any one of SEQ ID NOs: 8-10. In certain embodiments, substitutions, insertions or deletions occur in the outer region of the HVR (ie, FR). Optionally, the anti-DENV antibody comprises a VL sequence of any one of SEQ ID NO: 8-10, including post-translational modifications. In certain embodiments, the VL comprises an amino acid sequence of any one of (a) SEQ ID NO: 21-23, HVR-L1, and (b) SEQ ID NO: 24-26. Contains one, two, or three HVRs selected from HVR-L2 containing, and (c) SEQ ID NO: HVR-L3 containing any one amino acid sequence of 27-30. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 10. An anti-DENV antibody containing a light chain variable domain (VL) having the sequence identity of is provided. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to the reference sequence. An anti-DENV antibody containing a substitution (eg, conservative substitution), insertion, or deletion, but containing that sequence retains the ability to bind DENV. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and / or deleted at SEQ ID NO: 10. In certain embodiments, substitutions, insertions or deletions occur in the outer region of the HVR (ie, FR). Optionally, the anti-DENV antibody comprises a VL sequence of SEQ ID NO: 10, including post-translational modifications. In certain embodiments, the VL comprises (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 23, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and (c) SEQ ID NO. : Contains one, two, or three HVRs selected from HVR-L3 containing an amino acid sequence of 30. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In another aspect, an anti-DENV antibody comprising VH as in any of the above embodiments and VL as in any of the above embodiments is provided. In one aspect, the antibody comprises a VH sequence and a VL sequence of any one of SEQ ID NOs: 2-6 and any one of SEQ ID NOs 8-10, including post-translational modifications, respectively. In one aspect, the antibody comprises the VH and VL sequences of any one of SEQ ID NO: 2-6 and SEQ ID NO: 7, respectively, comprising post-translational modifications. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In another aspect, an anti-DENV antibody comprising VH as in any of the above embodiments and VL as in any of the above embodiments is provided. In one aspect, the antibody comprises a VH sequence and a VL sequence of SEQ ID NO: 6 and SEQ ID NO: 10, respectively, comprising post-translational modifications. In one aspect, the antibody comprises a VH sequence and a VL sequence of SEQ ID NO: 6 and SEQ ID NO: 7, respectively, comprising post-translational modifications. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-DENV antibody provided herein. In certain embodiments, at least one, at least two, at least three, at least four, selected from the group consisting of G100, W101, K122, I162, S274, K310, W391 and F392 on the DENV-2 E protein. Antibodies that bind to epitopes containing at least 5, at least 6, at least 7, or all amino acids are provided. In certain embodiments, an antibody that binds to an epitope comprising at least one, at least two, or all amino acids selected from the group consisting of K122, I162, and S274 on the DENV-2 E protein is provided. In certain embodiments, if the epitope contains at least one, at least two, or all amino acids selected from the group consisting of K122, I162, and S274, then from the group consisting of G100, W101, K310, W391, and F392. Antibodies that bind to the epitope further comprising at least one amino acid of choice are provided.

In one aspect, the invention provides a method of treating or preventing Zika virus infection. In one aspect, the invention provides a method of blocking the transmission of Zika virus from a pregnant mother to the foetation. In one aspect, the invention provides a method of preventing congenital Zika syndrome (eg, congenital dysgenesis, microcephaly, etc.). In one aspect, the invention provides a method of suppressing the loss of fetal weight (eg, whole body, head, etc.).

In one example, the term "congenital Zika syndrome" refers to a distinct pattern of birth defects typical of prenatally infected fetuses and newborns. As will be appreciated by those skilled in the art, subjects with congenital deca syndrome may exhibit symptoms such as, but not limited to: congenital dysgenesis, microcephaly, partial skull. Severe microcephaly that collapses, loss of brain tissue with certain patterns of brain damage such as subcortical calcification, damage to the fundus such as speckled scars and localized pigmented retinal spots, varus foot Or congenital contractions such as joint contractions, hypertonia that limits physical activity immediately after birth, etc. Administration of the antibodies described herein is believed to prevent at least one (or at least two, or at least three, or at least four, or all) symptoms of congenital deca syndrome. For example, the antibodies described herein prevent fetal or neonatal infection of prenatal Zika virus, thereby suppressing fetal weight loss (eg, systemic, head, etc.).

In some embodiments, the method of treating or preventing Zika virus infection of the present invention comprises administering an antibody that binds to Zika virus. In some embodiments, the method of blocking the transmission of Zika virus from a pregnant mother to the foetation comprises administering an antibody that binds to Zika virus. In some embodiments, the method of preventing congenital Zika syndrome (eg, congenital developmental deficiency, microcephaly, etc.) of the present invention comprises administering an antibody that binds to Zika virus. In some embodiments, the method of suppressing the loss of fetal weight (eg, whole body, head, etc.) of the present invention comprises administering an antibody that binds to Zika virus. In one example, the term "congenital Zika syndrome" refers to a distinct pattern of birth defects typical of prenatally infected fetuses and newborns. As will be appreciated by those skilled in the art, subjects with congenital deca syndrome may exhibit symptoms such as, but not limited to: congenital dysgenesis, microcephaly, partial skull. Severe microcephaly that collapses, loss of brain tissue with certain patterns of brain damage such as subcortical calcification, damage to the fundus such as speckled scars and localized pigmented retinal spots, varus foot Or congenital contractions such as joint contractions, hypertonia that limits physical activity immediately after birth, etc. Administration of the antibodies described herein is believed to prevent at least one (or at least two, or at least three, or at least four, or all) symptoms of congenital deca syndrome. For example, the antibodies described herein prevent fetal or neonatal infection of prenatal Zika virus, thereby suppressing fetal weight loss (eg, systemic, head, etc.).

In some embodiments, the antibody that binds to the Zika virus of the invention is cross-reactive with respect to dengue virus.

(ここでX₁は荷電アミノ酸であり、X₂は荷電アミノ酸または疎水性アミノ酸であり、X₃は荷電アミノ酸または疎水性アミノ酸である)を含む、HVR-H3、
(ii)アミノ酸配列：QQFX₁X₂LPIT(ここでX₁は荷電アミノ酸または極性アミノ酸であり、X₂は荷電アミノ酸または極性アミノ酸である)を含む、HVR-L3、および
(iii)アミノ酸配列：

(ここでX₁は荷電アミノ酸または極性アミノ酸であり、X₂は荷電アミノ酸または疎水性アミノ酸である)を含む、HVR-H2；
(b) (i)アミノ酸配列：SX₁YX₂H(ここでX₁は極性アミノ酸であり、X₂は疎水性アミノ酸である)を含む、HVR-H1、
(ii)アミノ酸配列：

(ここでX₁は荷電アミノ酸または極性アミノ酸であり、X₂は荷電アミノ酸または疎水性アミノ酸である)を含む、HVR-H2、および
(iii)アミノ酸配列：

(ここでX₁は荷電アミノ酸であり、X₂は荷電アミノ酸または疎水性アミノ酸であり、X₃は荷電アミノ酸または疎水性アミノ酸である)を含む、HVR-H3；
(c) (i)アミノ酸配列：SX₁YX₂H(ここでX₁は極性アミノ酸であり、X₂は疎水性アミノ酸である)を含む、HVR-H1、
(ii)アミノ酸配列：

(ここでX₁は荷電アミノ酸または極性アミノ酸であり、X₂は疎水性アミノ酸または荷電アミノ酸である)を含む、HVR-H2、
(iii)アミノ酸配列：

(ここでX₁は荷電アミノ酸であり、X₂は荷電アミノ酸または疎水性アミノ酸であり、X₃は荷電アミノ酸または疎水性アミノ酸である)を含む、HVR-H3、
(iv)アミノ酸配列：

(ここでX₁は荷電アミノ酸であり、X₂は荷電アミノ酸または極性アミノ酸である)を含む、HVR-L1、
(v)アミノ酸配列：DASX₁LKX₂(ここでX₁は荷電アミノ酸または極性アミノ酸であり、X₂は極性アミノ酸または疎水性アミノ酸である)を含む、HVR-L2、および
(vi)アミノ酸配列：QQFX₁X₂LPIT(ここでX₁は荷電アミノ酸または極性アミノ酸であり、X₂は荷電アミノ酸または疎水性アミノ酸である)を含む、HVR-L3；または
(d) (i)アミノ酸配列：

(ここでX₁は荷電アミノ酸であり、X₂は荷電アミノ酸または極性アミノ酸である)を含む、HVR-L1、
(ii)アミノ酸配列：DASX₁LKX₂(ここでX₁は極性アミノ酸または荷電アミノ酸であり、X₂は極性アミノ酸または疎水性アミノ酸である)を含む、HVR-L2、および
(iii)アミノ酸配列：QQFX₁X₂LPIT(ここでX₁は荷電アミノ酸または極性アミノ酸であり、X₂は荷電アミノ酸または疎水性アミノ酸である)を含む、HVR-L3。 In some embodiments, the antibody that binds to the Zika virus of the invention comprises:
(a) (i) Amino acid sequence:

(Where X ₁ is a charged amino acid, X ₂ is a charged or hydrophobic amino acid, and X ₃ is a charged or hydrophobic amino acid), HVR-H3,
(ii) Amino Acid Sequence: _{HVR-L3, which contains QQFX 1} X ₂ LPIT (where X ₁ is a charged or polar amino acid and X ₂ is a charged or polar amino acid), and
(iii) Amino acid sequence:

(Here X ₁ is a charged or polar amino acid and X ₂ is a charged or hydrophobic amino acid), HVR-H2;
(b) (i) Amino acid sequence: _{HVR-H1, which contains SX 1} YX ₂ H (where X ₁ is a polar amino acid and X ₂ is a hydrophobic amino acid),
(ii) Amino acid sequence:

HVR-H2, which contains (where X ₁ is a charged or polar amino acid and X ₂ is a charged or hydrophobic amino acid), and
(iii) Amino acid sequence:

(Where X ₁ is a charged amino acid, X ₂ is a charged or hydrophobic amino acid, and X ₃ is a charged or hydrophobic amino acid), HVR-H3;
(c) (i) Amino acid sequence: _{HVR-H1, which contains SX 1} YX ₂ H (where X ₁ is a polar amino acid and X ₂ is a hydrophobic amino acid),
(ii) Amino acid sequence:

HVR-H2, which contains (where X ₁ is a charged or polar amino acid and X ₂ is a hydrophobic or charged amino acid),
(iii) Amino acid sequence:

(Where X ₁ is a charged amino acid, X ₂ is a charged or hydrophobic amino acid, and X ₃ is a charged or hydrophobic amino acid), HVR-H3,
(iv) Amino acid sequence:

(Where X ₁ is a charged amino acid and X ₂ is a charged or polar amino acid), HVR-L1,
(v) Amino Acid Sequence: _{HVR-L2, which contains DASX 1} LKX ₂ (where X ₁ is a charged or polar amino acid and X ₂ is a polar or hydrophobic amino acid), and
(vi) Amino Acid Sequence: _{HVR-L3; or containing QQFX 1} X ₂ LPIT, where X ₁ is a charged or polar amino acid and X ₂ is a charged or hydrophobic amino acid.
(d) (i) Amino acid sequence:

(Where X ₁ is a charged amino acid and X ₂ is a charged or polar amino acid), HVR-L1,
(ii) Amino acid sequence: _{HVR-L2, which contains DASX 1} LKX ₂ (where X ₁ is a polar or charged amino acid and X ₂ is a polar or hydrophobic amino acid), and
(iii) Amino Acid Sequence: _{HVR-L3 comprising QQFX 1} X ₂ LPIT, where X ₁ is a charged or polar amino acid and X ₂ is a charged or hydrophobic amino acid.

As will be appreciated by those skilled in the art, charged amino acids include arginine (R, Arg), lycine (K, Lys), aspartic acid (D, Asp), and glutamic acid (E, Glu); polar amino acids. Glytamine (Q, Gln), aspartic acid (N, Asn), histidine (H, His), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine (C, Cys) ), And tryptophan (W, Trp); hydrophobic amino acids include alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe). , F), valine (Val, V), proline (Pro, P), and glycine (Gly, G).

(ここでX₁はRまたはEであり、X₂はRまたはFであり、X₃はG、DまたはLである)(SEQ ID NO: 42)を含む、HVR-H3、
(ii)アミノ酸配列：QQFX₁X₂LPIT(ここでX₁はD、SまたはEであり、X₂はDまたはAである)(SEQ ID NO: 45)を含む、HVR-L3、および
(iii)アミノ酸配列：

(ここでX₁はTまたはRであり、X₂はAまたはRである)(SEQ ID NO: 41)を含む、HVR-H2；
(b) (i)アミノ酸配列：SX₁YX₂H(ここでX₁はNまたはYであり、X₂はIまたはMである)(SEQ ID NO: 40)を含む、HVR-H1、
(ii)アミノ酸配列：

(ここでX₁はTまたはRであり、X₂はAまたはRである)(SEQ ID NO: 41)を含む、HVR-H2、および
(iii)アミノ酸配列：

(ここでX₁はRまたはEであり、X₂はRまたはFであり、X₃はG、DまたはLである)(SEQ ID NO: 42)を含む、HVR-H3；
(c) (i)アミノ酸配列：SX₁YX₂H(ここでX₁はNまたはYであり、X₂はIまたはMである)(SEQ ID NO: 40)を含む、HVR-H1、
(ii)アミノ酸配列：

(ここでX₁はTまたはRであり、X₂はAまたはRである)(SEQ ID NO: 41)を含む、HVR-H2、
(iii)アミノ酸配列：

(ここでX₁はRまたはEであり、X₂はRまたはFであり、X₃はG、DまたはLである)(SEQ ID NO: 42)を含む、HVR-H3、
(iv)アミノ酸配列：

(ここでX₁はDまたはEであり、X₂はKまたはQである)(SEQ ID NO: 43)を含む、HVR-L1、
(v)アミノ酸配列：DASX₁LKX₂(ここでX₁はNまたはEであり、X₂はTまたはFである)(SEQ ID NO: 44)を含む、HVR-L2、および
(vi)アミノ酸配列：QQFX₁X₂LPIT(ここでX₁はD、SまたはEであり、X₂はDまたはAである)(SEQ ID NO: 45)を含む、HVR-L3；または
(d) (i)アミノ酸配列：

(ここでX₁はDまたはEであり、X₂はKまたはQである)(SEQ ID NO: 43)を含む、HVR-L1、
(ii)アミノ酸配列：DASX₁LKX₂(ここでX₁はNまたはEであり、X₂はTまたはFである)(SEQ ID NO: 44)を含む、HVR-L2、および
(iii)アミノ酸配列：QQFX₁X₂LPIT(ここでX₁はD、SまたはEであり、X₂はDまたはAである)(SEQ ID NO: 45)を含む、HVR-L3。 In some embodiments, the antibody that binds to the Zika virus of the invention comprises:
(a) (i) Amino acid sequence:

(Where X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L) (SEQ ID NO: 42), HVR-H3,
(ii) Amino acid sequence: _{HVR-L3, including QQFX 1} X ₂ LPIT (where X ₁ is D, S or E, X ₂ is D or A) (SEQ ID NO: 45), and
(iii) Amino acid sequence:

(Here X ₁ is T or R and X ₂ is A or R) (SEQ ID NO: 41), HVR-H2;
(b) (i) Amino acid sequence: SX ₁ YX ₂ H (where X ₁ is N or Y and X ₂ is I or M) (SEQ ID NO: 40), HVR-H 1,
(ii) Amino acid sequence:

(Where X ₁ is T or R and X ₂ is A or R) (SEQ ID NO: 41), HVR-H2, and
(iii) Amino acid sequence:

(Where X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L) (SEQ ID NO: 42), HVR-H3;
(c) (i) Amino acid sequence: SX ₁ YX ₂ H (where X ₁ is N or Y and X ₂ is I or M) (SEQ ID NO: 40), HVR-H 1,
(ii) Amino acid sequence:

(Where X ₁ is T or R and X ₂ is A or R) (SEQ ID NO: 41), HVR-H2,
(iii) Amino acid sequence:

(Where X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L) (SEQ ID NO: 42), HVR-H3,
(iv) Amino acid sequence:

(Where X ₁ is D or E and X ₂ is K or Q) (SEQ ID NO: 43), HVR-L1,
(v) Amino acid sequence: _{HVR-L2, including DASX 1} LKX ₂ (where X ₁ is N or E and X ₂ is T or F) (SEQ ID NO: 44), and
(vi) Amino acid sequence: _{HVR-L3; or containing QQFX 1} X ₂ LPIT (where X ₁ is D, S or E and X ₂ is D or A) (SEQ ID NO: 45).
(d) (i) Amino acid sequence:

(Where X ₁ is D or E and X ₂ is K or Q) (SEQ ID NO: 43), HVR-L1,
(ii) Amino acid sequence: _{HVR-L2, including DASX 1} LKX ₂ (where X ₁ is N or E and X ₂ is T or F) (SEQ ID NO: 44), and
(iii) Amino acid sequence: _{HVR-L3 comprising QQFX 1} X ₂ LPIT (where X ₁ is D, S or E and X ₂ is D or A) (SEQ ID NO: 45).

In some embodiments, the antibody that binds to the Zika virus of the invention comprises:
(a) (i) HVR-H3 containing amino acid sequence SEQ ID NO: 20, (ii) HVR-L3 containing amino acid sequence SEQ ID NO: 27, and (iii) HVR-L3 containing amino acid sequence SEQ ID NO: 15 H2;
(b) (i) HVR-H1 containing the amino acid sequence SEQ ID NO: 12, (ii) HVR-H2 containing the amino acid sequence SEQ ID NO: 15, and (iii) HVR-H containing the amino acid sequence SEQ ID NO: 20. H3;
(c) (i) HVR-H1 containing amino acid sequence SEQ ID NO: 12, (ii) HVR-H2 containing amino acid sequence SEQ ID NO: 15, (iii) HVR-H3 containing amino acid sequence SEQ ID NO: 20 , (Iv) HVR-L1 containing amino acid sequence SEQ ID NO: 21, (v) HVR-L2 containing amino acid sequence SEQ ID NO: 24, and (vi) HVR-L3 containing amino acid sequence SEQ ID NO: 27; or
(d) (i) HVR-L1 containing amino acid sequence SEQ ID NO: 21, (ii) HVR-L2 containing amino acid sequence SEQ ID NO: 24, and (iii) HVR-L containing amino acid sequence SEQ ID NO: 27 L3.

In some embodiments, the antibodies of the invention are HVR-H1, which comprises the amino acid sequence of (i) SEQ ID NO: 11, HVR-H2, which comprises the amino acid sequence of (ii) SEQ ID NO: 13, and (iii) SEQ. HVR-H3 containing the amino acid sequence of ID NO: 16, HVR-L1 containing the amino acid sequence of (iv) SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of (v) SEQ ID NO: 24, and (vi) ) SEQ ID NO: Not an antibody containing HVR-L3 containing the amino acid sequence of 27.

In some embodiments, the antibody that binds to the Zika virus of the invention comprises:
(a) (i) SEQ ID NO: HVR-H3 from any one VH sequence of 2, 3, 4, 5, or 6, (ii) SEQ ID NO: any one of 8, 9, or 10. HVR-L3 from one VL sequence, and (iii) SEQ ID NO: HVR-H2 from any one VH sequence of 2, 3, 4, 5, or 6;
(b) (i) HVR-H1, (ii) SEQ ID NO: 2, 3, 4, 5, or 6 from any one VH sequence of SEQ ID NO: 2, 3, 4, 5, or 6. HVR-H2 from any one VH sequence of, and (iii) SEQ ID NO: HVR-H3 from any one VH sequence of 2, 3, 4, 5, or 6;
(c) (i) SEQ ID NO: 2, 3, 4, 5, or 6 HVR-H1, (ii) SEQ ID NO: 2, 3, 4, 5, or 6 from any one VH sequence HVR-H2 from any one VH sequence, (iii) SEQ ID NO: 2, 3, 4, 5, or 6 HVR-H3 from any one VH sequence, (iv) SEQ ID NO: HVR-L1, (v) SEQ ID NO: HVR-L2 from any one VL sequence of 8, 9, or 10 and (vi) SEQ ID NO: HVR-L2 from any one VL sequence of 8, 9, or 10. ID NO: HVR-L3 from any one VL sequence of 8, 9, or 10;
(d) HVR-L1 from any one VL sequence of (i) SEQ ID NO: 8, 9, or 10; (ii) from any one VL sequence of SEQ ID NO: 8, 9, or 10. HVR-L2, and (iii) HVR-L3 from any one VL sequence of SEQ ID NO: 8, 9, or 10; or
(e) (i) SEQ ID NO: HVR-H1, (ii) SEQ ID NO: 2, 3, 4, 5, or 6 from any one VH sequence of 2, 3, 4, 5, or 6. HVR-H2 from any one VH sequence, (iii) SEQ ID NO: 2, 3, 4, 5, or 6 HVR-H3 from any one VH sequence, (iv) SEQ ID NO: HVR-L1 from any one VL sequence of 7, (v) HVR-L2 from any one VL sequence of SEQ ID NO: 7, and VL of any one of (vi) SEQ ID NO: 7. HVR-L3 from the array.

In some embodiments, the antibodies of the invention are (i) HVR-H1 from the VH sequence of SEQ ID NO: 1, (ii) HVR-H2 from the VH sequence of SEQ ID NO: 1, and (iii) SEQ. HVR-H3 from VH sequence with ID NO: 1, (iv) HVR-L1 from VL sequence with SEQ ID NO: 7, (v) HVR-L2 from VL sequence with SEQ ID NO: 7, and (vi) ) SEQ ID NO: Not an antibody containing HVR-L3 from the VL sequence of 7.

In some embodiments, the antibody that binds to the dicavirus of the invention is FR1, which comprises the amino acid sequence of SEQ ID NO: 31 of the heavy chain variable domain framework, FR2, which comprises the amino acid sequence of SEQ ID NO: 32, SEQ ID. It further comprises FR3 containing the amino acid sequence of NO: 33 or 34, and FR4 containing the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibody that binds to the dicavirus of the invention is FR1, which comprises the amino acid sequence of SEQ ID NO: 36 of the light chain variable domain framework, FR2, which comprises the amino acid sequence of SEQ ID NO: 37. It further comprises FR3 containing the amino acid sequence of NO: 38 and FR4 containing the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the antibody that binds the dicavirus of the invention comprises: (a) SEQ ID NO: at least 95 for any one amino acid sequence of 2, 3, 4, 5, or 6. VH sequence with% sequence identity; (b) SEQ ID NO: VL sequence with at least 95% sequence identity to any one amino acid sequence of 8, 9, or 10; (c) SEQ ID NO: VH sequence of any one of 2, 3, 4, 5, or 6 and VL sequence of any one of SEQ ID NO: 8, 9, or 10; or (d) SEQ ID NO: 2, 3, A VH sequence of any one of 4, 5, or 6 and a VL sequence of any one of SEQ ID NO: 7.

In another aspect, the anti-ZIKV antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 for any one amino acid sequence of SEQ ID NO: 2-6. Includes heavy chain variable domain (VH) sequences with%, 98%, 99%, or 100% sequence identity. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to the reference sequence. The anti-ZIKV antibody containing the sequence, including substitution (eg, conservative substitution), insertion or deletion, retains the ability to bind ZIKV. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in any one of SEQ ID NOs: 2-6. In certain embodiments, substitutions, insertions or deletions occur in the outer region of the HVR (ie, FR). Optionally, the anti-ZIKV antibody comprises the VH sequence of any one of SEQ ID NO: 2-6, including post-translational modifications of that sequence. In certain embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and (c) SEQ ID NO. : Contains one, two, or three HVRs selected from HVR-H3s containing 20 amino acid sequences. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In another aspect, the anti-ZIKV antibody is at least 96%, 97%, 98%, 99%, or 100% for any one amino acid sequence of SEQ ID NO: 2, 3, 4, 5, or 6. Contains heavy chain variable domain (VH) sequences with sequence identity of. In certain embodiments, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (eg, conservative substitution), insertion or deletion with respect to the reference sequence. However, the anti-ZIKV antibody containing that sequence retains the ability to bind ZIKV. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted at any one of SEQ ID NOs: 2, 3, 4, 5, or 6. In certain embodiments, substitutions, insertions or deletions occur in the outer region of the HVR (ie, FR). Optionally, the anti-DENV antibody comprises a VH sequence of any one of SEQ ID NO: 2, 3, 4, 5, or 6 and comprises a post-translational modification of that sequence. In certain embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and (c) SEQ ID NO. : Contains one, two, or three HVRs selected from HVR-H3s containing 20 amino acid sequences. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In another aspect, an anti-ZIKV antibody is provided, which is at least 95%, 96%, 97%, 98%, 99 for any one amino acid sequence of SEQ ID NO: 8, 9, or 10. Includes a light chain variable domain (VL) with% or 100% sequence identity. In certain embodiments, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (eg, conservative substitution), insertion or deletion with respect to the reference sequence. However, the anti-ZIKV antibody containing that sequence retains the ability to bind DENV. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted at any one of SEQ ID NO: 8, 9, or 10. In certain embodiments, substitutions, insertions or deletions occur in the outer region of the HVR (ie, FR). Optionally, the anti-ZIKV antibody comprises a VL sequence of any one of SEQ ID NO: 8, 9, or 10 and comprises a post-translational modification of that sequence. In certain embodiments, the VL comprises (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 24, and (c) SEQ ID NO. : Contains one, two, or three HVRs selected from HVR-L3 containing an amino acid sequence of 27. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In another aspect, an anti-ZIKV antibody is provided, which has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. Includes a light chain variable domain (VL) with. In certain embodiments, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (eg, conservative substitution), insertion or deletion with respect to the reference sequence. However, the anti-ZIKV antibody containing that sequence retains the ability to bind ZIKV. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and / or deleted at SEQ ID NO: 10. In certain embodiments, substitutions, insertions or deletions occur in the outer region of the HVR (ie, FR). Optionally, the anti-DENV antibody comprises a VL sequence of SEQ ID NO: 10 and comprises a post-translational modification of that sequence. In certain embodiments, the VL comprises (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 24, and (c) SEQ ID NO. : Contains one, two, or three HVRs selected from HVR-L3 containing an amino acid sequence of 27. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In another aspect, an anti-ZIKV antibody is provided, which comprises the VH described in any of the embodiments provided above, and the VL described in any of the embodiments provided above. In one aspect, the antibody comprises VH and VL sequences of any one of SEQ ID NO: 2, 3, 4, 5, or 6 and any one of SEQ ID NO: 8, 9, or 10, respectively. , Includes post-translational modifications of these sequences. In one aspect, the antibody comprises the VH and VL sequences of any one of SEQ ID NO: 2, 3, 4, 5, or 6 and SEQ ID NO: 7, respectively, with post-translational modifications of these sequences. include. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In another aspect, an anti-ZIKV antibody is provided, which comprises the VH described in any of the embodiments provided above, and the VL described in any of the embodiments provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and comprises post-translational modifications of these sequences. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and comprises post-translational modifications of these sequences. Post-translational modifications include, but are not limited to, modification of heavy or light chain N-terminal glutamine or glutamate to pyroglutamyl by pyroglutamylation.

In some embodiments, the antibody comprises a constant heavy chain (ie, CH) sequence of SEQ ID NO: 59. In some embodiments, the antibody comprises a constant light chain (ie, CL) sequence of SEQ ID NO: 60. In some embodiments, the antibody comprises a constant heavy chain and a constant light chain sequence of SEQ ID NO: 59 and SEQ ID NO: 60, respectively.

In some embodiments, the antibody that binds to the Zika virus of the invention is a monoclonal antibody. In some embodiments, the antibody that binds to the Zika virus of the invention is a human antibody, a humanized antibody, or a chimeric antibody. In some aspects. The antibody that binds to the Zika virus of the present invention is a full-length IgG antibody.

In some embodiments, the Fc region of the antibody that binds to the Zika virus of the invention comprises Ala at position 234 and Ala at position 235 according to EU numbering. In some embodiments, the Fc region of the antibody that binds to the Zika virus of the invention can be selected from the mutant Fc regions described herein.

The present invention also provides a pharmaceutical preparation comprising the anti-dica antibody of the present invention and a pharmaceutically acceptable carrier.

The anti-dica antibody of the present invention may be intended for use as a pharmaceutical. In some embodiments, the anti-dica antibody of the invention may be for use in the treatment or prevention of deer infection.

The anti-dica antibody of the present invention can be used in the manufacture of pharmaceuticals. In some embodiments, the drug is for the treatment or prevention of deca infections.

The present invention also provides a method of treating an individual suffering from a deer infection. In some embodiments, the method comprises administering to the individual an effective amount of an anti-dica antibody of the invention. In some embodiments, the method further comprises administering to the individual an additional therapeutic agent, eg, as described below.

In some embodiments, the mutant Fc region of an antibody that binds to the Zika virus of the invention comprises at least one amino acid modification in the parent Fc region. In a further aspect, the mutant Fc region has a substantially reduced FcγR binding activity when compared to the parent Fc region. In a further aspect, the mutant Fc region does not have substantially reduced C1q binding activity when compared to the parent Fc region.

In some embodiments, the mutant Fc region of the antibody that binds to the Zika virus of the invention comprises Ala at position 234 and Ala at position 235 according to EU numbering. In a further aspect, the mutant Fc region comprises an amino acid modification of any one of (a)-(c) below: (a) positions 267, 268, and 324, (b) 236, 267, 268, 324. , And 332, and (c) 326 and 333; follow EU numbering.

In some embodiments, the mutant Fc region of the antibody that binds the Zika virus of the invention comprises an amino acid selected from the group consisting of: (a) Glu at position 267, (b) Phe at position 268, (c) Thr at 324, (d) Ala at 236, (e) Glu at 332, (f) Ala at 326, Asp, Glu, Met or Trp, and (g) Ser at 333; EU Follow the numbering.

In some embodiments, the mutant Fc region of the antibody that binds to the Zika virus of the invention further comprises an amino acid selected from the group consisting of: (a) Ala at position 434, (b) Ala at position 434: , 436th Thr, 438th Arg, and 440th Glu, (c) 428th Leu, 434th Ala, 436th Thr, 438th Arg, and 440th Glu, and (d) Follow EU numbering: Leu at 428, Ala at 434, Arg at 438, and Glu at 440.

In some embodiments, the mutant Fc region of an antibody that binds to the Zika virus of the invention comprises any of the amino acid modifications, alone or in combination, as described in Table 4. In some embodiments, the parent Fc region described in the present invention is derived from human IgG1. The present invention provides a polypeptide comprising any one amino acid sequence of SEQ ID NO: 51-59.

In a further aspect, the antibody that binds to the Zika virus of the invention comprises:
(a) (i) HVR-H3 containing the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 27, and (iii) the amino acid sequence of SEQ ID NO: 13. Including HVR-H2;
(b) HVR-H1 containing the amino acid sequence of (i) SEQ ID NO: 11, HVR-H2 containing the amino acid sequence of (ii) SEQ ID NO: 13, and (iii) the amino acid sequence of SEQ ID NO: 16. Including HVR-H3;
(c) (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 13, and (iii) containing the amino acid sequence of SEQ ID NO: 16. HVR-H3, HVR-L1 containing the amino acid sequence of (iv) SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of (v) SEQ ID NO: 24, and the amino acid sequence of (vi) SEQ ID NO: 27 Including HVR-L3; or
(d) (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) the amino acid sequence of SEQ ID NO: 27. Including HVR-L3;
(e) (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 12, (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) the amino acid sequence of SEQ ID NO: 20. Including HVR-H3.

In a further aspect, the antibody that binds to the Zika virus of the invention comprises:
(a) (i) HVR-H3 from the VH sequence of SEQ ID NO: 6, (ii) HVR-L3 from the VL sequence of SEQ ID NO: 10, and (iii) from the VH sequence of SEQ ID NO: 6. HVR-H2;
(b) (i) HVR-H1 from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 from the VH sequence of SEQ ID NO: 6, and (iii) from the VH sequence of SEQ ID NO: 6. HVR-H3;
(c) (i) HVR-H1 derived from VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from VH sequence of SEQ ID NO: 6, (iii) derived from VH sequence of SEQ ID NO: 6. HVR-H3, HVR-L1 from the VL sequence of (iv) SEQ ID NO: 10, HVR-L2 from the VL sequence of (v) SEQ ID NO: 10, and the VL sequence of (vi) SEQ ID NO: 10. Origin HVR-L3;
(d) (i) HVR-L1 from the VL sequence of SEQ ID NO: 10, (ii) HVR-L2 from the VL sequence of SEQ ID NO: 10, and (iii) from the VL sequence of SEQ ID NO: 10. HVR-L3; or
(e) (i) HVR-H1 derived from VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from VH sequence of SEQ ID NO: 6, (iii) derived from VH sequence of SEQ ID NO: 6. HVR-H3, HVR-L1 from the VL sequence of (iv) SEQ ID NO: 7, HVR-L2 from the VL sequence of (v) SEQ ID NO: 7, and the VL sequence of (vi) SEQ ID NO: 7. Origin HVR-L3.

In a further aspect, the antibody that binds to the dicavirus of the invention is (i) VH comprising the amino acid sequence of SEQ ID NO: 6, (ii) VL comprising the amino acid sequence of SEQ ID NO: 7, and (iii) SEQ ID. Contains CH containing the amino acid sequence of NO: 59 and CL containing the amino acid sequence of (iv) SEQ ID NO: 60.

In a further aspect, antibodies that bind to the dicavirus of the invention are from SG182 (WT; SEQ ID NO: 46), SG1095 (SEQ ID NO: 54) and SG1106 (3Cam2-LALA + KAES + ACT5 CH; SEQ ID NO: 59). VH variant sequence selected from the group consisting of 3CH1047 (3Cam2-LALA + KAES + ACT5 VH; SEQ ID NO: 6) and 3CH1049 (SEQ ID NO: 95) having a human IgG1 CH sequence selected from the group; VL selected from the group consisting of 3CL (VL; SEQ ID NO: 7) and 3CL633 (SEQ ID NO: 98) with CL sequence SK1 (CL of 3Cam2-LALA + KAES + ACT5; SEQ ID NO: 60) Contains variant sequences. The variants present and the sequences of the mutations are shown and detailed in Tables 2 and 4.

In some examples, antibodies that bind the Zika virus of the invention are exemplified in the "Examples" section of the present disclosure. For example, the antibodies described herein can be the antibodies disclosed in Example 7, eg, but not limited to, the following variants: (a) 3Cam2, which is DG_3CH1047 (SEQ). ID NO: 6)-SG182 (SEQ ID NO: 46) / 3CL (SEQ ID NO: 7) _SK1 (SEQ ID NO: 60); (b) 3Cam2-LALA + KAES, which is DG_3CH1047 (SEQ ID NO) : 6)-SG1095 (SEQ ID NO: 54) / 3CL (SEQ ID NO: 7) _SK1 (SEQ ID NO: 60); (c) 3Cam2-LALA + KAES + ACT5, which is DG_3CH1047 (SEQ ID NO) : 6)-SG1106 (SEQ ID NO: 59) / 3CL (SEQ ID NO: 7) _SK1 (SEQ ID NO: 60); (d) 3C, which is DG_3CH (SEQ ID NO: 1)-SG182 ( SEQ ID NO: 46) / 3CL (SEQ ID NO: 7) _SK1 (SEQ ID NO: 60); (e) 3C-LALA, which is DG_3CH (SEQ ID NO: 1) -SG192 (SEQ ID NO:: 47) / 3CL (SEQ ID NO: 7) _SK1 (SEQ ID NO: 60); (e) 3C-LALA + KAES + ACT5, which is DG_3CH (SEQ ID NO: 1)-SG1106 (SEQ ID NO:: 59) / 3CL (SEQ ID NO: 7) _SK1 (SEQ ID NO: 60); and similar.

In a further aspect of the invention, the anti-DENV antibody or anti-ZIKV antibody according to any of the above embodiments is a monoclonal antibody, such as a chimeric antibody, a humanized antibody, a human antibody. In one aspect, the anti-DENV or anti-ZIKV antibody is an antibody fragment, eg, Fv, Fab, Fab', scFv, diabody, or F (ab') ₂ fragment. In another aspect, the antibody is a full-length antibody, eg, an intact IgG1 antibody, or another antibody class or isotype as defined herein.

In a further aspect, the anti-DENV or anti-ZIKV antibodies of the invention may have modifications that negate the binding of the antibody to the Fc gamma receptor (FcγR). Without being bound by theory, modifications that abolish antibody binding to the Fc gamma receptor suggest that reduced binding to FcR is primarily mediated by interaction with FcR. It can be advantageous because it can avoid the ADE (antibody-dependent enhancement) phenomenon. In one aspect, the Fc region of the anti-DENV antibody of the invention comprises Ala at position 234 and Ala at position 235 according to EU numbering.

In a further aspect, the anti-DENV antibody or anti-ZIKV antibody according to any of the above embodiments may incorporate any of the features described in items 1-7 below, alone or in combination.

1. 1. Antibody Affinity In certain embodiments, the antibodies provided herein are 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less or 0.001 nM or less (eg, ^10-8 M or less, It has a dissociation constant (Kd) of, for example, 10 ^{-8 M to 10 -13} M, for example 10 ^{-9 M to 10 -13 M).}

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one aspect, RIA is performed with the Fab version of the antibody of interest and its antigen. For example, Fab's in-solution binding affinity for ^{antigen equilibrates Fab with the lowest concentration of (125} I) labeled antigen in the presence of an increasing series of unlabeled antigens, and then coats the bound antigen with an anti-Fab antibody. Measured by capturing with an antigen. (See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the measurement conditions, a MICROTITER® multiwell plate (Thermo Scientific) was coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by Block with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23 ° C). On a non-adsorbed plate (Nunc # 269620), 100 pM or 26 pM [ ¹²⁵ I] -antigen (eg, Presta et al., Cancer Res. 57: 4593-4599 (1997) anti-VEGF antibody, Fab- Mix with the serial dilution of Fab of interest (similar to evaluation 12). The Fab of interest is then incubated overnight, which can be continued for a longer period of time (eg, about 65 hours) to ensure equilibrium is achieved. The mixture is then transferred to a capture plate for incubation at room temperature (eg, 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 in PBS (TWEEN-20®). Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20 ™, Packard) and count the plate on a TOPCOUNT ™ gamma counter (Packard) for 10 minutes. The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

According to another aspect, Kd is measured using the BIACORE® surface plasmon resonance assay. For example, an assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is a CM5 chip with approximately 10 response unit (RU) antigens immobilized. Is carried out at 25 ° C. In one embodiment, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is N-ethyl-N'-(3-dimethylaminopropyl) -carbodiimidehydrochloride (EDC) and N according to the supplier's instructions. -Activated with hydroxysuccinimide (NHS). Antigen to 5 μg / ml (approximately 0.2 μM) using 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve approximately 10 reaction unit (RU) protein binding. It is diluted. After injection of the antigen, 1M ethanolamine is injected to block unreacted groups. 2-fold serial dilutions of Fab in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20 ™) detergent at 25 ° C. and a flow rate of approximately 25 μl / min for reaction rate measurements. ~ 500nM) is injected. Binding rate (k _on ) and dissociation rate (k _off ) are determined by simultaneously fitting the binding and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). It is calculated. The equilibrium dissociation constant (Kd) is calculated as the _{k off} / k _{on ratio.} See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). ^{If the on-velocity exceeds 10 6} M ^-1 s ^-1 by the surface plasmon resonance assay described above, the on-velocity is the 8000 series SLM- using a spectrometer (eg, a stop-flow spectrophotometer (Aviv Instruments) or a stirring cuvette). Fluorescence intensity (excitation) of 20 nM anti-antigen antibody (Fab form) at 25 ° C in PBS, pH 7.2 in the presence of increasing concentrations of antigen as measured by an AMINCO ™ spectrophotometer (ThermoSpectronic). = 295 nm; emission = 340 nm, bandpass 16 nm) can be determined using fluorescence quenching techniques that measure the increase or decrease.

2. Antibody Fragments In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F (ab') ₂ , Fv, and scFv fragments, as well as other fragments described below. See Hudson et al. Nat. Med. 9: 129-134 (2003) for a review of specific antibody fragments. As a review of scFv fragments, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); 16185; and see US Pat. Nos. 5,571,894 and 5,587,458. See U.S. Pat. No. 5,869,046 for an editorial on _{Fab and F (ab') 2} fragments with extended half-lives in vivo containing salvage receptor binding epitope residues.

A diabody is an antibody fragment with two antigen binding sites, which may be divalent or bispecific. For example, EP404,097; WO1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) ) reference. Triabody and tetrabody are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

A single domain antibody is an antibody fragment that comprises all or part of the heavy chain variable domain of an antibody, or all or part of the light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody (see Domantis, Inc., Waltham, MA; eg, US Pat. No. 6,248,516 B1).

Antibody fragments vary, including, but not limited to, the proteolytic digestion of complete antibodies described herein, production by recombinant host cells (eg, E. coli or phage). It can be made by the method of.

3. 3. Chimeric and Humanized Antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Specific chimeric antibodies are described, for example, in US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, the chimeric antibody comprises a non-human variable region (eg, a variable region derived from a non-human primate such as mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, the chimeric antibody is a "class switch" antibody whose class or subclass has changed from that of the parent antibody. Chimeric antibodies also include their antigen-binding fragments.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while preserving the specificity and affinity of the parent non-human antibody. Usually, a humanized antibody comprises one or more variable domains, in which the HVR (eg CDR (or portion thereof)) is derived from a non-human antibody and the FR (or portion thereof) is in the human antibody sequence. Derived from. The humanized antibody optionally comprises at least a portion of the human constant region. In some embodiments, some FR residues in a humanized antibody are, for example, the antibody from which the non-human antibody (eg, the HVR residue was derived, to restore or improve the specificity or affinity of the antibody. ) Is replaced with the corresponding residue.

Humanized antibodies and methods of their preparation are reviewed in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008) and, for example, Riechmann et al., Nature 332: 323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); US Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36 : 25-34 (2005) (specificity determining region (SDR) graphing described); Padlan, Mol. Immunol. 28: 489-498 (1991) (described "resurfacing"); Dall 'Acqua et al., Methods 36: 43-60 (2005) (described "FR shuffling"); as well as Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Further described in Cancer, 83: 252-260 (2000) (described "guide selection" approach for FR shuffling).

The human framework regions that can be used for humanization are not limited to these: selected using the "best fit" method (see Sims et al. J. Immunol. 151: 2296 (1993)). Framework Regions; Framework Regions Derived from the Consensus Sequence of Human Antibodies in Specific Subgroups of Light Chain or Heavy Chain Variable Regions (Carter et al. Proc. Natl. Acad. Sci. USA 89: 4285 (1992) and Presta et al. J. Immunol. 151: 2623 (1993)); Human maturation (somatic mutation) framework region or human germ cell system framework region (eg, Almagro and Fransson, Front. Biosci. 13: 1619-1633) (See (2008)); and framework regions derived from FR library screening (Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (see 1996)).

4. Human Antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by a variety of techniques known in the art. Human antibodies are outlined in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal modified to produce a fully human antibody or a fully antibody with a human variable region in response to an antigen challenge. Such animals typically contain all or part of the human immunoglobulin locus, and all or part of the human immunoglobulin locus replaces the endogenous immunoglobulin locus, or extrachromosomally or It exists in a state of being randomly incorporated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. See Lonberg, Nat. Biotech. 23: 1117-1125 (2005) for a review of how to obtain human antibodies from transgenic animals. Also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE ™ technology; U.S. Patent No. 5,770,429 describing HUMAB ™ technology; U.S. Patent No. 5 describing KM MOUSE ™ technology. See also US Patent Application Publication No. 7,041,870; and US Patent Application Publication No. 2007/0061900, which describes VELOCIMOUSE® technology. Human variable regions from complete antibodies produced by such animals may be further modified, for example by combining with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already been described. (For example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al. ., J. Immunol. 147: 86 (1991).) Human antibodies produced via human B cell hybridoma technology are also Li et al., Proc. Natl. Acad. Sci. USA 103: 3557-3562 ( It is stated in 2006). Additional methods include, for example, US Pat. No. 7,189,826 (described for the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) (humans). -Includes those described in (listing human hybridomas). Human hybridoma technology (trioma technology) is also Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3): 185-91. It is described in (2005).

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from the human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domain. The method for selecting human antibodies from the antibody library is described below.

5. Library-Derived Antibodies The antibodies of the invention may be isolated by screening a combinatorial library for antibodies with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are reviewed in Hoogenboom et al. In Methods in Molecular Biology 178: 1-37; O'Brien et al., Ed., Human Press, Totowa, NJ, 2001), and further, for example, McCafferty. et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al., J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al. , J. Immunol. Methods 284 (1-2): 119-132 (2004).

In a particular phage display method, the repertoire of VH and VL genes is cloned separately by the polymerase chain reaction (PCR) and randomly recombined in the phage library, which winter et al. It can be screened for antigen-binding phage as described in., Ann. Rev. Immunol. 12: 433-455 (1994). Phage typically present an antibody fragment, either as a single chain Fv (scFv) fragment or as a Fab fragment. Libraries from immunized sources provide high affinity antibodies to the immune source without the need to build hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), the naive repertoire is cloned (eg, from humans) and broadly non-self and without immunization. It is also possible to provide a single source of antibodies to self-antigens. Finally, the naive library cloned the pre-reorganized V-gene segment from stem cells and hypervariable CDR3 as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). It can also be made synthetically by using PCR primers that encode the region and contain random sequences to achieve in vitro reconstruction. Patent documents describing the human antibody phage library include, for example: US Pat. No. 5,750,373, and US Patent Application Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007. Includes / 0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

An antibody or antibody fragment isolated from a human antibody library is considered herein as a human antibody or human antibody fragment.

6. Multispecific Antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (eg, bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificity at at least two different sites. In certain embodiments, one of the binding specificities is for the DENVE protein and the other is for any other antigen. In certain embodiments, the bispecific antibody may bind to two different epitopes of the DENVE protein. Bispecific antibodies may be used to localize cytotoxic agents in cells expressing the DENVE protein. Bispecific antibodies can be prepared as full-length antibodies or as antibody fragments.

Techniques for making multispecific antibodies are not limited to these, but are recombinant co-expressions of two immunoglobulin heavy chain-light chain pairs with different specificities (Milstein and Cuello, Nature 305: 537). (1983), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" technology (see, eg, US Pat. No. 5,731,168). Multispecific antibodies manipulate electrostatic steering effects to create Fc heterodimer molecules (WO 2009/089004A1); crosslink two or more antibodies or fragments (USA). See Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); Using a leucine zipper to produce antibodies with two specificities (Kostelny et al., J. Immunol. 148 (5). ): 1547-1553 (1992)); Making bispecific antibody fragments using the "diabody" technique (Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 () (See 1993); and use a single-chain Fv (scFv) dimer (see Gruber et al., J. Immunol. 152: 5368 (1994)); and, for example, Tutt et al., J. Immunol. It may be made by preparing a trispecific antibody as described in 147: 60 (1991).

Modified antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006/0025576 A1).

As used herein, an antibody or fragment also includes a "dual acting Fab" or "DAF" that comprises one antigen binding site that binds the DENVE protein to another different antigen (eg, US Patent Application Publication No. 2008. / See No. 0069820).

7. Antibody Variants In certain embodiments, the amino acid sequence variants of the antibodies provided herein are also considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion of the antibody from the amino acid sequence and / or insertion of the antibody into the amino acid sequence and / or substitution of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to reach the final construct, provided that the final construct has the desired characteristics (eg, antigen binding).

a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown under the heading "Favorable substitutions" in Table 1. More substantive changes are provided under the "Exemplary Substitution" heading in Table 1 and are detailed below with reference to the class of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest and the product may be screened for the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. good.

(Table 1)

Amino acids can be grouped according to common side chain properties:
(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;
(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;
(3) Acidity: Asp, Glu;
(4) Basicity: His, Lys, Arg;
(5) Residues affecting chain orientation: Gly, Pro;
(6) Aromatic: Trp, Tyr, Phe.
Non-conservative permutation refers to exchanging a member of one of these classes for a member of another class.

One type of substitution variant involves substitution of one or more hypervariable region residues of the parent antibody (eg, humanized or human antibody). Usually, the resulting variants selected for further study are modified (eg, improved) (eg, increased affinity, decreased immunogenicity) in certain biological properties compared to the parent antibody. ) And / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity maturation antibody, which can be appropriately made, for example, using phage display-based affinity maturation techniques (eg, those described herein). Briefly, one or more HVR residues are mutated and the mutated antibody is presented on the phage to screen for specific biological activity (eg, binding affinity).

Modifications (eg, substitutions) can be made, for example, in HVR to improve antibody affinity. Such modifications are the "hot spots" of HVR, that is, residues encoded by codons that frequently mutate during the somatic cell maturation process (eg, Chowdhury, Methods Mol. Biol. 207: 179-196). (See (2008)) and / or at residues in contact with the antigen, and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. In Methods in Molecular Biology 178: 1-37 (O'Brien et al., Ed., Human Press, Totowa, NJ, (2001). )) It is described in. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any variety of methods (eg, error-prone PCR, chain shuffling or oligonucleotide-directed mutagenesis). Next, a secondary library is created. The library is then screened to identify any antibody variant with the desired affinity. Another way to introduce diversity involves an HVR-oriented approach that randomizes several HVR residues (eg, 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions can be made within one or more HVRs, as long as such modifications do not substantially reduce the ability of the antibody to bind the antigen. For example, conservative modifications that do not substantially reduce binding affinity (eg, conservative substitutions as provided herein) can be made in the HVR. Such modifications can be, for example, outside the antigen contact residues of HVR. In certain embodiments of the above mutant VH and VL sequences, each HVR is unmodified or contains only one, two, or three amino acid substitutions.

A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is described by Cunningham and Wells (1989), Science, 244: 1081-1085, "Alanine scanning mutagenesis". It is called. In this method, a residue or group of target residues (eg, charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified and neutral or negatively charged amino acids (eg, alanine or). It is replaced with polyalanine) to determine if the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that are functionally sensitive to this initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be analyzed to identify the point of contact between the antibody and the antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or excluded from substitution candidates. Mutants can be screened to determine if they contain the desired properties.

The insertion of an amino acid sequence, similar to the insertion of a single or multiple amino acid residues inside the sequence, is in the length range of the polypeptide containing 1 to 100 or more residues at the amino and / or carboxyl terminus. Including fusion. An example of a terminal insertion comprises an antibody with a methionyl residue at the N-terminus. Other insertion variants of the antibody molecule include an enzyme (eg, for ADEPT) or a polypeptide that increases the plasma half-life of the antibody fused to the N- or C-terminus of the antibody.

b) Glycosylated variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be easily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

If the antibody contains an Fc region, the carbohydrate added to it may be modified. Native antibodies produced by mammalian cells typically contain branched bifurcated oligosaccharides, which are usually added by N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15: 26-32 (1997). Oligosaccharides include various carbohydrates such as, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose added to GlcNAc in the "stem" of the bifurcated oligosaccharide structure. In some embodiments, modifications of oligosaccharides in the antibodies of the invention may be made to produce antibody variants with certain improved properties.

In one aspect, an antibody variant having a carbohydrate structure lacking fucose added (directly or indirectly) to the Fc region is provided. For example, the amount of fucose in such antibodies can be 1% -80%, 1% -65%, 5% -65% or 20% -40%. The amount of fucose is measured by MALDI-TOF mass spectrometry, eg, WO 2008/077546, for all sugar structures added to Asn297 (eg, complex, hybrid, and high mannose structures). Determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum. Asn297 represents an asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to the slight sequence diversity between multiple antibodies, Asn297 can also be located approximately +/- 3 amino acids upstream or downstream at position 297, ie between positions 294-300. Such fucosylated variants may have improved ADCC function. See, for example, US Patent Application Publication No. 2003/0157108 (Presta, L.); No. 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications on "defucosylated" or "fucos-deficient" antibody variants are US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Includes Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004). An example of a cell line capable of producing a defucosylated antibody is a Lec13 CHO cell lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Patent Application Publication No. US 2003/0157108 A1, Presta, L; and WO 2004/056312A1, Adams et al., Especially Example 11) and knockout cell lines such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (eg Yamane). -Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda Y. et al., Biotechnol. Bioeng. 94 (4): 680-688 (2006); and WO 2003/085107) include.

For example, an antibody variant having a bisected oligosaccharide in which the bifurcated oligosaccharide added to the Fc region of the antibody is bisected by GlcNAc is further provided. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umana et al.); And US2005 / 0123546 (Umana et al.). There is. Antibody variants with at least one galactose residue in the oligosaccharide added to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc region variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (eg, the Fc region of human IgG1, IgG2, IgG3, or IgG4) that comprises an amino acid modification (eg, substitution) at one or more amino acid positions.

In certain embodiments, antibody variants having some, but not all, effector functions are also within consideration of the present invention, where the effector function is such that the half-life of the antibody in vivo is important. Certain effector functions (such as ADCC) are good candidates for application when they are unnecessary or harmful. In vitro and / or in vivo cytotoxicity measurements can be performed to measure CDC and / or ADCC activity. For example, Fc receptor (FcR) binding measurements can be performed to determine if an antibody has FcγR binding (and thus is more likely to have ADCC activity) and / or FcRn binding. NK cells, which are the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). A non-limiting example of an in vitro assay for assessing ADCC activity of a molecule of interest is US Pat. No. 5,500,362 (eg, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83: 7059- 7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); US Pat. No. 5,821,337 (Bruggemann, M. et al., J. Exp) . Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays may be used (eg, ACT1 ™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96 ™ non-radioactive cytotoxicity assays. Law (see Promega, Madison, WI)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest is assessed in vivo in an animal model such as that described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). May be done. In addition, C1q binding measurement may be performed to confirm whether the antibody can bind to C1q and therefore has CDC activity. See, for example, C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be made to assess complement activation (eg, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101). 1045-1052 (2003); and Cragg, MS and MJ Glennie Blood 103: 2738-2743 (2004)). In addition, FcRn binding and in vivo clearance / half-life determinations can also be made using methods known in the art (eg, Petkova, et al., Int'l. Immunol. 18 (12): 1759). -1769 (2006)).

Antibodies with modified effector function include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Pat. No. 6,737,056). Such Fc variants include so-called "DANA" Fc variants (US Pat. No. 7,332,581) with substitutions of residues 265 and 297 for alanine at amino acid positions 265, 269, 270, 297, and 327. Includes Fc variants with two or more substitutions.

Specific antibody variants with modified binding to FcR have been described. (See U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001).)

In certain embodiments, the antibody variant undergoes one or more amino acid substitutions that modify ADCC (eg, substitutions at positions 298, 333, and / or 334 (EU numbering residues) of the Fc region). Includes the accompanying Fc region.

In some embodiments, it has been modified (ie, as described, for example, in US Pat. No. 6,194,551, WO99 / 51642, WO2011 / 091078, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000). Modifications that result in C1q binding and / or complement-dependent cytotoxicity (CDC) are made in the Fc region.

Increased half-life and neonatal Fc receptors (FcRn: responsible for translocating maternal IgGs to the fetal (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Antibodies with increased binding to Immunol. 24: 249 (1994))) are described in US Patent Application Publication No. 2005/0014934 A1 (Hinton et al.). These antibodies include an Fc region with one or more substitutions therein that increase the binding of the Fc region to FcRn. Such Fc variants include Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382. , 413, 424, or 434 with one or more substitutions (eg, substitution of Fc region residue 434 (US Pat. No. 7,371,826)).

See also Duncan & Winter, Nature 322: 738-40 (1988); US Pat. No. 5,648,260 and US Pat. No. 5,624,821; and WO 94/29351 for other examples of Fc region variants.

In another embodiment, the antibody may comprise a mutant Fc of the invention detailed herein below.

d) Cysteine-modified antibody variants In certain embodiments, it may be desirable to produce a cysteine-modified antibody, such as "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the residue to be substituted occurs at an accessible site of the antibody. By substituting those residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, and the reactive thiol groups attach the antibody to other moieties (drug moiety or linker-drug moiety). Etc.) and may be used to create an immunoconjugate as described in more detail herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: light chain V205 (Kabat numbering); heavy chain A118 (EU numbering); and heavy chain Fc region S400. (EU numbering). Cysteine-modified antibodies may be produced, for example, as described in US Pat. No. 7,521,541.

e) Antibody Derivatives In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers are, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-Dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (either homopolymer or random copolymer), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol Includes homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde would be advantageous in production due to its stability to water. The polymer may have any molecular weight and may or may not be branched. The number of polymers added to the antibody may vary, and they may be the same molecule or different molecules as long as one or more polymers are added. In general, the number and / or type of polymer used for derivatization is not limited to these, but the particular property or function of the antibody to be improved, the antibody derivative under specified conditions. It can be decided based on consideration such as whether or not it is used for therapy.

In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). Radiation can be of any wavelength, and is not limited to these, such as heating the non-protein portion to a temperature that does not harm normal cells but kills cells in close proximity to the antibody-non-protein portion. Including wavelength.

In one aspect, the invention provides an isolated polypeptide containing a mutant Fc region with substantially reduced FcγR binding activity. In one aspect, the invention provides an isolated polypeptide containing a mutant Fc region that does not have substantially reduced C1q binding activity. In one aspect, the invention provides an isolated polypeptide comprising a mutant Fc region that has substantially reduced FcγR binding activity and no substantially reduced C1q binding activity. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the mutant Fc region has at least one amino acid residue compared to the corresponding sequence of the Fc region of the native sequence or reference variant sequence (collectively referred to herein as the "parent" Fc region). Includes base modification (eg, substitution). In certain embodiments, the mutant Fc region of the invention has a substantially reduced FcγR binding activity as compared to the parent Fc region. In certain embodiments, the mutant Fc region of the invention does not have substantially reduced C1q binding activity as compared to the parent Fc region. In certain embodiments, the FcγR is a human FcγR, a monkey FcγR (eg, cynomolgus monkey, rhesus monkey, marmoset, chimpanzee, or baboon FcγR), or a mouse FcγR.

In one aspect, the mutant Fc region of the invention is compared to the parent Fc region, eg, FcγRIa, FcγRIIa (including allelic variants 167H and 167R), FcγRIIb, FcγRIIIa (including allelic variants 158F and 158V). , And FcγRIIIb, including but not limited to allelic variants NA1 and NA2, have substantially reduced binding activity to one or more human FcγRs. In a further aspect, the mutant Fc regions of the invention are compared to human FcγRIa, FcγRIIa (including allelic variants 167H and 167R), FcγRIIb, FcγRIIIa (including allelic variants 158F and 158V), And have substantially reduced binding activity to FcγRIIIb, including allelic variants NA1 and NA2.

In one aspect, the mutant Fc region of the invention was substantially reduced for one or more mouse FcγRs including, but not limited to, FcγRI, FcγRIIb, FcγRIII, and FcγRIV as compared to the parent Fc region. Has binding activity. In a further aspect, the mutant Fc region of the invention has substantially reduced binding activity to mouse FcγRI, FcγRIIb, FcγRIII, and FcγRIV as compared to the parent Fc region.

"Fcγ receptor" (referred to herein as Fcγ receptor, FcγR or FcgR) refers to a receptor capable of binding to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and is actually an Fcγ receptor. Means any member of a family of proteins encoded by a body gene. In humans, this family includes, but is not limited to: FcγRI (CD64), eg isoforms FcγRIa, FcγRIb, FcγRIc, etc .; FcγRII (CD32), eg isoform FcγRIIa (allotype H131 (type H) and R131 (including type R), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), FcγRIIc, etc .; and FcγRIII (CD16), eg isoforms FcγRIIIa (including allotypes V158 and F158), FcγRIIIb (including allotype FcγRIIIb-) (Including NA1 and FcγRIIIb-NA2); and human FcγR, FcγR isoforms or allotypes that have not yet been discovered. FcγRIIb-1 and FcγRIIb-2 have been reported as splicing variants of human FcγRIIb. In addition, a splicing mutant named FcγRIIb-3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splicing variants, human FcγRIIb contains all splicing variants registered with NCBI, which are NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992. It is 3. In addition, human FcγRIIb includes all previously reported genetic polymorphisms, not just FcγRIIb (Arthritis Rheum. 48: 3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14: 2881. -2892 (2005); and Kyogoju et al., Arthritis Rheum. 46: 1242-1254 (2002)), and all future reported genetic polymorphisms are included.

There are two allotypes of FcγRIIa, one in which the amino acid at position 167 of FcγRIIa is histidine (type H) and the other in which the amino acid at position 167 is replaced with arginine (type R) (Warrmerdam,). J. Exp. Med. 172: 19-25 (1990)).

FcγR includes, but is not limited to, FcγR derived from humans, mice, rats, rabbits, and monkeys, and may be derived from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIV (CD16-2), as well as any mouse FcγR, or FcγR isoform.

The amino acid sequence of human FcγRIa is shown in SEQ ID NO: 69; the amino acid sequence of human FcγRIIa (167H) is shown in SEQ ID NO: 70; the amino acid sequence of human FcγRIIa (167R) is shown in SEQ ID NO: 71. The amino acid sequence of human FcγRIIb is shown in SEQ ID NO: 72; the amino acid sequence of human FcγRIIIa (158F) is shown in SEQ ID NO: 73; the amino acid sequence of human FcγRIIIa (158V) is shown in SEQ ID NO: 74. The amino acid sequence of human FcγRIIIb (NA1) is shown in SEQ ID NO: 75; the amino acid sequence of human FcγRIIIb (NA2) is shown in SEQ ID NO: 76.

The amino acid sequence of mouse FcγRI is shown in SEQ ID NO: 77; the amino acid sequence of mouse FcγRIIb is shown in SEQ ID NO: 78; the amino acid sequence of mouse FcγRIII is shown in SEQ ID NO: 79; the amino acid sequence of mouse FcγRIV is shown. Is shown in SEQ ID NO: 80.

In one aspect, the variant Fc region of the invention has a substantially reduced FcγR binding activity, which is less than 50%, less than 45%, as a function of the FcγR binding activity of the parent Fc region. Less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or 0.1 Less than%. In one aspect, the mutant Fc region of the invention has a substantially reduced FcγR binding activity, which indicates that the RU value of the sensorgram changed before and after the interaction of FcγR with the mutant Fc region. Difference] / [Difference in RU value of sensorgram changed before and after capturing FcγR on sensor chip] ratio is less than 1, less than 0.8, less than 0.5, less than 0.3, less than 0.2, less than 0.1, less than 0.08, less than 0.05, It means less than 0.03, less than 0.02, less than 0.01, less than 0.008, less than 0.005, less than 0.003, less than 0.002, or less than 0.001.

In one aspect, the variant Fc region of the invention does not have a substantially reduced C1q binding activity, which is due to the difference in C1q binding activity between the variant Fc region of the invention and the parent Fc region. , Less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or 5 as a function of C1q binding activity in the parent Fc region Means less than%.

In one aspect, the invention provides an isolated polypeptide containing a mutant Fc region with substantially reduced ADCC activity. In one aspect, the invention provides an isolated polypeptide containing a mutant Fc region that does not have substantially reduced CDC activity. In one aspect, the invention provides an isolated polypeptide comprising a mutant Fc region having substantially reduced ADCC activity and not substantially reduced CDC activity.

In one aspect, the mutant Fc region of the invention is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20% as a function of ADCC activity of the parent Fc region. Has substantially reduced ADCC activity, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1%.

In one aspect, the variant Fc region of the invention does not have substantially reduced CDC activity, which is due to the difference in CDC activity between the variant Fc region of the invention and the parent Fc region. As a function of CDC activity in the Fc region, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% It means that there is.

In one aspect, the invention simply comprises a mutant Fc region that has a substantially reduced FcγR binding activity and does not have a substantially reduced C1q binding activity as compared to a polypeptide containing a parent Fc region. The separated polypeptide is provided. In a further aspect, the polypeptides of the invention are modified with at least one amino acid at at least one position selected from the group consisting of 234, 235, 236, 267, 268, 324, 326, 332, and 333 according to EU numbering. including.

In one aspect, the mutant Fc region with substantially reduced FcγR binding activity and not substantially reduced C1q binding activity follows Ala at position 234, Ala at position 235, and according to EU numbering. Includes at least one amino acid modification at at least one position selected from the group consisting of 236, 267, 268, 324, 326, 332, and 333.

In one aspect, the mutant Fc region with substantially reduced FcγR binding activity and not substantially reduced C1q binding activity follows Ala at position 234, Ala at position 235, and according to EU numbering. Includes further amino acid modification of any one of (a)-(c) below: (a) positions 267, 268, and 324; (b) positions 236, 267, 268, 324, and 332; and (c). 326 and 333th.

In a further aspect, the mutant Fc region with substantially reduced FcγR binding activity and not substantially reduced C1q binding activity is (a) at position 267 Glu; (b) at position 268 according to EU numbering. Phe; (c) Thr at 324; (d) Ala at 236; (e) Glu at 332; (f) Ala, Asp, Glu, Met, or Trp; and (g) 333 at 326. Contains amino acids selected from the group consisting of Ser.

In one aspect, the mutant Fc region with substantially reduced FcγR binding activity and not substantially reduced C1q binding activity contains the following amino acids according to EU numbering: Ala at position 234, position 235. Ala, 326th Ala, and 333th Ser. In one aspect, the mutant Fc region with substantially reduced FcγR binding activity and not substantially reduced C1q binding activity contains the following amino acids according to EU numbering: Ala at position 234, position 235. Ala, Asp at 326, and Ser at 333. In one aspect, the mutant Fc region with substantially reduced FcγR binding activity and not substantially reduced C1q binding activity contains the following amino acids according to EU numbering: Ala at position 234, position 235. Ala, Glu at 326, and Ser at 333. In one aspect, the mutant Fc region with substantially reduced FcγR binding activity and not substantially reduced C1q binding activity contains the following amino acids according to EU numbering: Ala at position 234, position 235. Ala, Met at 326th, and Ser at 333th. In one aspect, the mutant Fc region with substantially reduced FcγR binding activity and not substantially reduced C1q binding activity contains the following amino acids according to EU numbering: Ala at position 234, position 235. Ala, Trp at 326, and Ser at 333.

In another aspect, the mutant Fc region of the invention may further comprise at least one amino acid modification at at least one position selected from the group consisting of 428, 434, 436, 438, and 440 according to EU numbering. can.

In a further aspect, the mutant Fc region can further contain amino acids selected from the group consisting of: (a) Ala at position 434; (b) Ala at position 434, at position 436, according to EU numbering. Thr, Arg at 438, and Glu at 440; (c) Leu at 428, Ala at 434, Thr at 436, Arg at 438, and Glu at 440; and (d) Leu at 428. , Ala at position 434, Arg at position 438, and Glu at position 440 (see also WO2016 / 125495, which describes the relationship between amino acid modification and FcRn binding activity of the mutant Fc region).

In another aspect, the mutant Fc region of the invention contains the following amino acids according to EU numbering: Ala at position 234, Ala at position 235, Ala at position 326, Ser at position 333, Leu at position 428, 434. Ala in rank, Thr in 436th, Arg in 438th, and Glu in 440th. In another aspect, the mutant Fc region of the invention contains the following amino acids according to EU numbering: Ala at position 234, Ala at position 235, Ala at position 326, Ser at position 333, Leu at position 428, 434. Ala in rank, Arg in 438th, and Glu in 440th.

In one aspect, it is preferred that the mutant Fc region of the invention does not have a substantially increased FcRn binding activity, especially at pH 7.4, as compared to the parent Fc region.

"FcRn" is structurally similar to major histocompatibility complex (MHC) class I polypeptides and exhibits 22% to 29% sequence identity with MHC class I molecules. FcRn is expressed as a heterodimer consisting of a soluble β chain or light chain (β2 microglobulin) complexed with a transmembrane α chain or heavy chain. Similar to MHC, the α chain of FcRn contains three extracellular domains (α1, α2, and α3), the short cytoplasmic domains that anchor them to the cell surface. The α1 and α2 domains interact with the FcRn-binding domain of the antibody Fc region. The polynucleotide and amino acid sequences of human FcRn can be derived, for example, from the precursors (including signal sequences) shown in NM_004107.4 and NP_004098.1, respectively.

The amino acid sequence of human FcRn (α chain) is shown in SEQ ID NO: 81; the amino acid sequence of human β2 microglobulin is shown in SEQ ID NO: 82.

In one aspect, the mutant Fc region of the invention is less than 1000-fold, less than 500-fold, less than 200-fold, less than 100-fold, less than 90-fold compared to the FcRn-binding activity of the parent Fc region, especially at pH 7.4. , Less than 80 times, less than 70 times, less than 60 times, less than 50 times, less than 40 times, less than 30 times, less than 20 times, less than 10 times, less than 5 times, less than 3 times, or less than 2 times, substantially It is preferable that it does not have an increased FcRn binding activity. In one aspect, the mutant Fc region of the invention does not have a substantially increased FcRn binding activity, especially at pH 7.4, which changes before and after the interaction of FcRn with the mutant Fc region. The ratio of [Difference in RU value of sensorgram] / [Difference in RU value of sensorgram changed before and after capturing FcRn on sensor chip] is less than 0.5, less than 0.3, less than 0.2, less than 0.1, less than 0.08, less than 0.05. , Less than 0.03, less than 0.02, less than 0.01, less than 0.008, less than 0.005, less than 0.003, less than 0.002, or less than 0.001.

In another aspect, the mutant Fc region of the invention comprises any of the amino acid modifications listed in Table 4, alone or in combination. In another aspect, the mutant Fc region of the invention comprises at least one of the amino acid modifications listed in Table 4. In another aspect, the invention provides a polypeptide comprising any one amino acid sequence of SEQ ID NO: 51-59.

In some embodiments, the polypeptide comprising the mutant Fc region of the invention is a constant region of the antibody heavy chain. In some embodiments, the polypeptide comprising the mutant Fc region of the invention further comprises an antigen binding domain. In a further aspect, the polypeptide is an antibody heavy chain. In a further aspect, the polypeptide is an antibody. In certain embodiments, the antibody is a chimeric antibody or a humanized antibody. The origin of the antibody is not particularly limited, and examples thereof include human antibody, mouse antibody, rat antibody, and rabbit antibody. In a further aspect, the polypeptide is an Fc fusion protein.

Two or more polypeptides containing the mutant Fc region described herein can be included in one molecule, in which case the two polypeptides containing the mutant Fc region are associated, like an antibody. Will be done. The type of antibody is not limited, and IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM and the like can be used.

In some embodiments, the polypeptide comprising the mutant Fc region of the invention is an antibody. In a further aspect, the polypeptide comprising the mutant Fc region of the invention is an antiviral antibody.

In a further aspect, the polypeptide comprising the mutant Fc region of the invention comprises an antibody variable region comprising:
(a) (i) HVR-H3 containing the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 27, and (iii) the amino acid sequence of SEQ ID NO: 13. Including HVR-H2;
(b) HVR-H1 containing the amino acid sequence of (i) SEQ ID NO: 11, HVR-H2 containing the amino acid sequence of (ii) SEQ ID NO: 13, and (iii) the amino acid sequence of SEQ ID NO: 16. Including HVR-H3;
(c) (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 13, and (iii) containing the amino acid sequence of SEQ ID NO: 16. HVR-H3, HVR-L1 containing the amino acid sequence of (iv) SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of (v) SEQ ID NO: 24, and the amino acid sequence of (vi) SEQ ID NO: 27 Including HVR-L3; or
(d) (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) the amino acid sequence of SEQ ID NO: 27. Including HVR-L3.

In a further aspect, the polypeptide comprising the mutant Fc region of the invention comprises an antibody variable region comprising:
(a) (i) HVR-H3 from the VH sequence of SEQ ID NO: 6, (ii) HVR-L3 from the VL sequence of SEQ ID NO: 10, and (iii) from the VH sequence of SEQ ID NO: 6. HVR-H2;
(b) (i) HVR-H1 from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 from the VH sequence of SEQ ID NO: 6, and (iii) from the VH sequence of SEQ ID NO: 6. HVR-H3;
(c) (i) HVR-H1 derived from VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from VH sequence of SEQ ID NO: 6, (iii) derived from VH sequence of SEQ ID NO: 6. HVR-H3, HVR-L1 from the VL sequence of (iv) SEQ ID NO: 10, HVR-L2 from the VL sequence of (v) SEQ ID NO: 10, and the VL sequence of (vi) SEQ ID NO: 10. Origin HVR-L3;
(d) (i) HVR-L1 from the VL sequence of SEQ ID NO: 10, (ii) HVR-L2 from the VL sequence of SEQ ID NO: 10, and (iii) from the VL sequence of SEQ ID NO: 10. HVR-L3; or
(e) (i) HVR-H1 derived from VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from VH sequence of SEQ ID NO: 6, (iii) derived from VH sequence of SEQ ID NO: 6. HVR-H3, HVR-L1 from the VL sequence of (iv) SEQ ID NO: 7, HVR-L2 from the VL sequence of (v) SEQ ID NO: 7, and the VL sequence of (vi) SEQ ID NO: 7. Origin HVR-L3.

In a further aspect, the polypeptide comprising the mutant Fc region of the present invention comprises a VH region comprising the amino acid sequence of SEQ ID NO: 1 and a VL region comprising the amino acid sequence of SEQ ID NO: 7. In a further aspect, the invention comprises a VH region containing the amino acid sequence of SEQ ID NO: 1 in the heavy chain and a mutant Fc region containing the amino acid sequence of SEQ ID NO: 54 in the heavy chain and an amino acid of SEQ ID NO: 7 in the light chain. An antibody containing a VL region containing a sequence is provided. In a further aspect, the invention comprises a VH region containing the amino acid sequence of SEQ ID NO: 1 in the heavy chain and a mutant Fc region containing the amino acid sequence of SEQ ID NO: 58 in the heavy chain and an amino acid of SEQ ID NO: 7 in the light chain. An antibody containing a VL region containing a sequence is provided. In a further aspect, the invention comprises a VH region containing the amino acid sequence of SEQ ID NO: 1 in the heavy chain and a mutant Fc region containing the amino acid sequence of SEQ ID NO: 59 in the heavy chain and an amino acid of SEQ ID NO: 7 in the light chain. An antibody containing a VL region containing a sequence is provided.

The present invention also provides the anti-DENV antibodies described herein further comprising a polypeptide comprising the mutant Fc region of the present invention. In some embodiments, the anti-DENV antibody of the invention has a VH region containing the amino acid sequence of SEQ ID NO: 6 on the heavy chain and a mutant Fc region containing the amino acid sequence of SEQ ID NO: 54 on the light chain and SEQ on the light chain. Contains the VL region containing the amino acid sequence of ID NO: 10. In some embodiments, the anti-DENV antibody of the invention has a VH region containing the amino acid sequence of SEQ ID NO: 6 on the heavy chain and a mutant Fc region containing the amino acid sequence of SEQ ID NO: 58 on the light chain. Contains the VL region containing the amino acid sequence of ID NO: 10. In some embodiments, the anti-DENV antibody of the invention has a VH region containing the amino acid sequence of SEQ ID NO: 6 on the heavy chain and a mutant Fc region containing the amino acid sequence of SEQ ID NO: 59 on the light chain and SEQ on the light chain. Contains the VL region containing the amino acid sequence of ID NO: 10.

In some embodiments, the anti-DENV antibody of the invention has a VH region containing the amino acid sequence of SEQ ID NO: 6 on the heavy chain and a mutant Fc region containing the amino acid sequence of SEQ ID NO: 59 on the light chain and SEQ on the light chain. Contains the VL region containing the amino acid sequence of ID NO: 7.

As used herein, the "parent Fc region" refers to the Fc region before the introduction of the amino acid modification (s) described herein. Preferred examples of the parent Fc region include an Fc region derived from a native antibody. Antibodies include IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM and the like. Antibodies can be of human or monkey origin (eg, cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons). Natural antibodies may contain naturally occurring mutations. "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, describes multiple allotype sequences of IgG due to genetic polymorphisms, all of which can be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356 to 358 (EU numbering) can be either DEL or EEM. Preferred examples of the parental Fc region are human IgG1 (SEQ ID NO: 83), human IgG2 (SEQ ID NO: 84), human IgG3 (SEQ ID NO: 85), and human IgG4 (SEQ ID NO: 86). The Fc region derived from the heavy chain constant region is included. Another preferred example of the parent Fc region is the Fc region from the heavy chain constant region SG1 (SEQ ID NO: 87). Another preferred example of the parent Fc region is the Fc region from the heavy chain constant region SG182 (SEQ ID NO: 46). Further, the parent Fc region may be an Fc region brought about by adding an amino acid modification (s) other than the amino acid modification described in the present specification to the Fc region derived from a natural antibody.

In addition, amino acid modifications made for other purposes can be combined in the mutant Fc regions described herein. For example, amino acid substitutions that improve FcRn binding activity (Hinton et al., J. Immunol. 176 (1): 346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281 (33): 23514-23524 (2006); Petkova et al., Intl. Immunol. 18 (12): 1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28 (2): 157-159 (2010); WO 2006/019447; WO 2006/053301; and WO 2009/086320), and amino acid substitutions to improve antibody heterogeneity or stability (WO 2009/041613) can be added. Alternatively, a polypeptide having the property of promoting antigen clearance (described in WO 2011/122011, WO 2012/132067, WO 2013/046704 or WO 2013/180201), or a polypeptide having the property of specifically binding to a target tissue (described in WO 2013/046704 or WO 2013/180201). WO 2013/180200), a polypeptide having the property of repeatedly binding to multiple antigen molecules (described in WO 2009/125825, WO 2012/073992 or WO 2013/047752) in the mutant Fc region described herein. Can be combined with. Alternatively, the amino acid modifications disclosed in EP1752471 and EP1772465 can be combined in CH3 of the mutant Fc region described herein for the purpose of conferring binding ability to other antigens. Alternatively, amino acid modifications (WO 2012/016227) that reduce pI in the constant region can be combined in the mutant Fc region described herein for the purpose of increasing plasma retention. Alternatively, amino acid modifications that increase pI in the constant region (WO 2014/145159) can be combined in the mutant Fc region described herein for the purpose of facilitating uptake into cells. Alternatively, amino acid modifications (WO2016 / 125495 and WO2016 / 098357) that increase pI in the constant region can be combined in the mutant Fc region described herein for the purpose of facilitating the excretion of the target molecule from plasma. ..

Amino acid modifications that enhance human FcRn binding activity under acidic pH can also be combined in the mutant Fc regions described herein. Specifically, such modifications include, for example: the substitution of Met at position 428 with Leu and the substitution of Asn at position 434 with Ser according to EU numbering (Nat Biotechnol, 2010, 28: 157). -159); Substitution of Asn at position 434 to Ala (Drug Metab Dispos, 2010 Apr; 38 (4): 600-605); Substitution of Met at position 252 to Tyr, Substitution of Ser at position 254 to Thr And the substitution of Thr at position 256 with Glu (J Biol Chem, 2006, 281: 23514-23524); the substitution of Thr at position 250 with Gln and the substitution of Met at position 428 with Leu (J Immunol, 2006, 176). (1): 346-356); Substitution of Asn at position 434 to His (Clin Pharmacol Ther, 2011, 89 (2): 283-290); and WO2010 / 106180, WO2010 / 045193, WO2009 / 058492, WO2008 / Modifications described in 022152, WO2006 / 050166, WO2006 / 053301, WO2006 / 031370, WO2005 / 123780, WO2005 / 047327, WO2005 / 037867, WO2004 / 035752, WO2002 / 060919, etc. In another aspect, such modifications are selected from the group consisting of, for example, the substitution of Met at position 428 with Leu, the substitution of Asn at position 434 with Ala, and the substitution of Tyr at position 436 with Thr. It can contain at least one modification. These modifications can further include the substitution of Gln at position 438 with Arg and / or the substitution of Ser at position 440 with Glu (WO2016 / 125495).

In the present invention, amino acid modification means any one of substitution, deletion, addition, insertion, and modification, or a combination thereof. In the present invention, amino acid modification can be paraphrased as amino acid mutation.

Amino acid modifications are brought about by various methods known to those of skill in the art. Such methods include site-directed mutagenesis (Hashimoto-Gotoh et al., Gene 152: 271-275 (1995); Zoller, Meth. Enzymol. 100: 468-500 (1983); Kramer et al. , Nucleic Acids Res. 12: 9441-9456 (1984)); Kramer and Fritz, Methods Enzymol. 154: 350-367 (1987); and Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985) )), PCR mutagenesis, and cassette mutagenesis, but are not limited to these.

The number of amino acid modifications introduced into the Fc region is not limited. In certain embodiments, it is 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 8 or less, 10 or less, 12 or less, 14 or less, 16 or less, 18 or less, or 20. It can be:

In addition, the polypeptides containing the mutant Fc region of the invention can be chemically modified with various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Chemical modification methods for such polypeptides have been established in the art.

In some embodiments, the polypeptide comprising the mutant Fc region of the invention is an antibody, or Fc fusion protein comprising a domain (s) capable of binding to any antigen. Examples of antigens that can be bound by such antibodies and Fc fusion proteins include, but are not limited to, ligands (cytokines, chemocaines, etc.), receptors, cancer antigens, viral antigens, MHC antigens, differentiation antigens, immunoglobulins. , And immune complexes that partially contain immunoglobulins.

B. Recombinant Methods and Compositions Antibodies can be made using, for example, the recombinant methods and compositions described in US Pat. No. 4,816,567. In one aspect, an isolated nucleic acid encoding the anti-DENV antibody described herein is provided. In one embodiment, the anti-DENV antibody is cross-reactive with ZIKV. In another embodiment, an isolated nucleic acid encoding a polypeptide comprising the mutant Fc region or parent Fc region described herein is provided. Such nucleic acids may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising VH (eg, the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors containing such nucleic acids (eg, expression vectors) are provided. In a further embodiment, a host cell containing such nucleic acid is provided. In such an embodiment, the host cell comprises (eg, transformed below): (1) a nucleic acid encoding an amino acid sequence comprising VL of the antibody and an amino acid sequence comprising VH of the antibody. , Vector, or (2) a first vector containing a nucleic acid encoding an amino acid sequence containing VL of an antibody, and (2) a second vector containing a nucleic acid encoding an amino acid sequence containing VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, eg, a Chinese hamster ovary (CHO) cell or a lymphocyte cell (eg, Y0, NS0, Sp2 / 0 cell). In one aspect, a method of making an anti-DENV antibody is provided, in which a host cell containing a nucleic acid encoding the antibody, as provided above, is cultured under conditions suitable for expression of the antibody. It comprises a step and optionally the step of recovering the antibody from the host cell (or host cell culture medium). In another embodiment, a method of making a polypeptide comprising a mutant Fc region or a parent Fc region is provided, the method encoding a polypeptide such as an antibody, Fc region, or mutant Fc region as provided above. It comprises a step of culturing a host cell containing the nucleic acid to be used under conditions suitable for expression of the polypeptide, and optionally, a step of recovering the polypeptide from the host cell (or host cell culture medium).

To recombinantly produce an anti-DENV antibody, one or more vectors for isolating the nucleic acid encoding the antibody, eg, as described above, for further cloning and / or expression in host cells. Insert into. To recombine the Fc region, the nucleic acid encoding the Fc region is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional techniques (eg, by using oligonucleotide probes capable of specifically binding the genes encoding the heavy and light chains of the antibody). ..

Suitable host cells for cloning or expression of the vector encoding the antibody include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, especially if glycosylation and Fc effector functions are not required. See, for example, US Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 for expression of antibody fragments and polypeptides in bacteria. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254, which describes the expression of antibody fragments in E. coli.) , Antibodies may be isolated in soluble fractions from bacterial cell paste and can be further purified.

Eukaryotes, such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways are "humanized", resulting in the production of antibodies with partial or complete human glycosylation patterns in addition to prokaryotes. Nuclear microorganisms are suitable cloning or expression hosts for antibody coding vectors. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

Those derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that are used for conjugation with insect cells, especially for transformation of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, US Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (described PLANTIBODIES ™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, a mammalian cell line adapted to grow in a suspended state would be useful. Another example of a useful mammalian host cell line is the SV40-transformed monkey kidney CV1 line (COS-7); the human fetal kidney line (Graham et al., J. Gen Virol. 36:59 (1977). ); 293 or 293 cells described in (1980), etc.; Mammal hamster kidney cells (BHK); Mather, Biol. Reprod. 23: 243-251 (1980), etc. ); African green monkey kidney cells (VERO-76); Human cervical cancer cells (HELA); Canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); Human lung cells (W138); Human hepatocytes ( Hep G2); mouse breast cancer (MMT 060562); TRI cells (eg, described in Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines are ^{Chinese hamster ovary (CHO) cells, including DHFR-} CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and Y0, Includes myeloma cell lines such as NS0, and Sp2 / 0. As a review of specific mammalian host cell lines suitable for antibody production, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268. See (2003).

Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Related antigens to proteins that are immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine succinimide, or soybeantrypsin inhibitor, bifunctional substances or derivatizers, such as , Maleimide benzoyl succinimide ester (conjugation via cysteine residue), N-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, SOCL ₂ , or R ¹ N = C = NR (where R) And R ¹ are different alkyl groups), it may be useful to conjugate.

Animals (usually non-human mammals) may, for example, inject 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) intradermally at multiple sites in combination with a triple volume Freund complete adjuvant. Is immunized against an antigen, immunogenic conjugate, or derivative. One month later, the animal is boosted with 1/5 to 1/10 of the initial amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, blood is drawn from the animal and the serum is assayed for antibody titers. Reimmunize the animal until the titer reaches the plateau. Preferably, the animal is boost-immunized with a conjugate of the same antigen but conjugated to another protein and / or via another cross-linking reagent. Conjugates can also be prepared as protein fusions in recombinant cell cultures. In addition, a flocculant such as alum is also preferably used to enhance the immune response.

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population may have a small amount of naturally occurring potential mutations and / or post-translational modifications (eg, eg). It is the same except for isomerization and amidation). Therefore, the modifier "monoclonal" indicates an antibody characteristic that it is not a mixture of distinct antibodies.

For example, monoclonal antibodies can be made using the hybridoma method first described in Kohler et al., Nature 256 (5517): 495-497 (1975). The hybridoma method has the ability to immunize a mouse or other suitable host animal, such as a hamster, as described herein to produce or produce an antibody that specifically binds to the protein used for immunization. Induces certain lymphocytes. Alternatively, lymphocytes can be immunized in vitro.

The immunizing agent typically comprises an antigenic protein or a fusion variant thereof. In general, peripheral blood lymphocytes (PBLs) are used when cells of human origin are desired, and spleen cells or lymph node cells are used when non-human mammalian sources are desired. Lymphocytes are then fused with the immortalized cell line using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59- 103).

Immortalized cell lines are usually transformed mammalian cells, especially rodent, bovine and human-derived myeloma cells. Usually, rat or mouse myeloma cell lines are utilized. The hybridoma cells thus prepared are seeded and proliferated in a suitable culture medium preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine annin phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas typically contains hypoxanthine, aminopterin, which are substances that interfere with the growth of HGPRT-deficient cells. And will contain thymidine (HAT medium).

Preferred immortalized myeloma cells are cells that fuse efficiently, assist in the stable, high-level production of antibodies by selected antibody-producing cells, and are sensitive to media such as HAT medium. .. Among these, mouse myeloma strains, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center in San Diego, Calif., And the American Type Culture Collection in Manassas, Virginia, USA. SP-2 cells (and derivatives thereof, such as X63-Ag8-653) are preferred. For the production of human monoclonal antibodies, human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described (Kozbor et al., J Immunol. 133 (6): 3001-3005 (1984); Brodeur et al. ., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

Culture medium in which hybridoma cells are growing is assayed for the production of monoclonal antibodies to the antigen. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson, Anal Biochem. 107 (1): 220-239 (1980).

After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity have been identified, the clones can be subcloned by limiting dilution and propagated by standard methods (Goding, supra). .. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also grow in vivo as tumors in mammals.

Monoclonal antibodies secreted by subclones are obtained from culture medium, ascites, or serum by conventional immunoglobulin purification methods such as, for example, protein A-cepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. , Properly separated.

The Fc region may be obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibody, etc. with a protease such as pepsin, and then re-eluting the fraction adsorbed on the protein A column. The protease is not particularly limited as long as it can digest the full-length antibody, thereby producing Fab and F (ab') 2 in a restrictive manner by appropriately setting enzymatic reaction conditions such as pH. , Examples include pepsin and papain.

Furthermore, the present invention produces a polypeptide containing a mutant Fc region with a substantially reduced FcγR binding activity as compared to a polypeptide containing a parent Fc region and without a substantially reduced C1p binding activity. The method comprises the step of introducing at least one amino acid modification into the parent Fc region. In some aspects, the polypeptide produced is an antibody. In certain embodiments, the antibody is a chimeric antibody or a humanized antibody. In some aspects, the polypeptide produced is an Fc fusion protein.

In one aspect, according to EU numbering, 234, in the above method for preparing a polypeptide comprising a variant Fc region having substantially reduced FcγR binding activity and not substantially reduced C1q binding activity, 234, At least one amino acid at at least one position selected from the group consisting of 235, 236, 267, 268, 324, 326, 332, and 333 is modified.

In another aspect, in the above method for preparing a polypeptide containing a mutant Fc region having a substantially reduced FcγR binding activity and not a substantially reduced C1q binding activity, the two amino acids are 234. It is modified at the place and the 235th place.

In another aspect, the amino acids are modified in the above method for preparing a polypeptide containing a variant Fc region having a substantially reduced FcγR binding activity and a substantially reduced C1q binding activity. The modifications include: according to EU numbering, selected from the group consisting of (a) two amino acid modifications at positions 234 and 235, and (b) 236, 267, 268, 324, 326, 332, and 333. At least one amino acid modification at at least one position.

In another aspect, the amino acids are modified in the above method for preparing a polypeptide containing a variant Fc region having a substantially reduced FcγR binding activity and a substantially reduced C1q binding activity. The modifications include: (a) two amino acid modifications at positions 234 and 235, and (b) at least one amino acid modification of any one of (i)-(iii) below, according to EU numbering: ( i) 267, 268, and 324; (ii) 236, 267, 268, 324, and 332; (iii) 326 and 333.

In a further aspect, the amino acid modification in the above method for preparing a polypeptide containing a mutant Fc region having a substantially reduced FcγR binding activity and not a substantially reduced C1q binding activity is performed at each position. , Selected from the group consisting of: (a) Ala at 234th place; (b) Ala at 235th place; (c) Glu at 267th place; (d) Phe at 268th place; (e) 324 Thr; (f) 236th Ala; (g) 332th Glu; (h) 326th Ala, Asp, Glu, Met, Trp; and (i) 333th Ser.

In a further aspect, the amino acid modification in the above method for preparing a polypeptide containing a mutant Fc region having a substantially reduced FcγR binding activity and not a substantially reduced C1q binding activity is according to EU numbering. , 234th Ala, 235th Ala, 326th Ala, and 333th Ser. In a further aspect, the amino acid modification in the above method for preparing a polypeptide containing a mutant Fc region having a substantially reduced FcγR binding activity and not a substantially reduced C1q binding activity is according to EU numbering. , 234th Ala, 235th Ala, 326th Asp, and 333th Ser. In a further aspect, the amino acid modification in the above method for preparing a polypeptide containing a mutant Fc region having a substantially reduced FcγR binding activity and not a substantially reduced C1q binding activity is according to EU numbering. , 234th Ala, 235th Ala, 326th Glu, and 333th Ser. In a further aspect, the amino acid modification in the above method for preparing a polypeptide containing a mutant Fc region having a substantially reduced FcγR binding activity and not a substantially reduced C1q binding activity is according to EU numbering. , 234th Ala, 235th Ala, 326th Met, and 333th Ser. In a further aspect, amino acid modifications in the above methods for preparing a polypeptide containing a variant Fc region with substantially reduced FcγR binding activity and not substantially reduced C1q binding activity are according to EU numbering. , 234th Ala, 235th Ala, 326th Trp, and 333th Ser.

In another aspect, in the above method, at least one amino acid is further modified at at least one position selected from the group consisting of 428, 434, 436, 438, and 440 according to EU numbering.

In a further aspect, the amino acid modification in the above method is further selected from (a)-(d) below: (a) Ala at position 434; (b) Ala at position 434, Thr at position 436, according to EU numbering. , 438th Arg, and 440th Glu; (c) 428th Leu, 434th Ala, 436th Thr, 438th Arg, and 440th Glu; and (d) 428th Leu, Ala at position 434, Arg at position 438, and Glu at position 440 (see also WO2016 / 125495, which describes the relationship between amino acid modification and FcRn binding activity of the mutant Fc region).

In a further aspect, the amino acid modifications in the above method, according to EU numbering, are Ala at position 234, Ala at position 235, Ala at position 326, Ser at position 333, Leu at position 428, Ala at position 434, Thr at position 436, Arg at 438th and Glu at 440th. In a further aspect, the amino acid modifications in the above method, according to EU numbering, are Ala at position 234, Ala at position 235, Ala at position 326, Ser at position 333, Leu at position 428, Ala at position 434, Arg at position 438, And 440th Glu.

In one aspect, it is preferred that the mutant Fc region of the invention does not have a substantially increased FcRn binding activity, especially at pH 7.4, as compared to the parent Fc region.

In a further aspect, the amino acid modification in the production method described above is selected from any single modification, a combination of single modifications, or a combination modification described in Table 4.

Polypeptides containing mutant Fc regions produced by any of the above methods or other methods known in the art are also included in the present invention.

C. Assays The anti-DENV antibodies provided herein have been identified, screened, or identified for physical / chemical properties and / or biological activity by various assays known in the art. You may.

Mutant Fc regions provided herein have been identified, screened, or identified for physical / chemical properties and / or biological activity by various assays known in the art. May be good.

1. 1. Binding Assays and Other Assays In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods such as ELISA, Western blot. In one aspect, the polypeptide containing the mutant Fc region of the invention is tested for its antigen binding activity by known methods such as ELISA, Western blot and the like.

In another aspect, competing assays can be used to identify antibodies that compete with any of the anti-DENV antibodies described herein for binding to DENV and / or DENVE protein. In certain embodiments, if there is an excess of such competing antibody, it is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, Blocks (eg, reduces) binding of reference antibodies to DENV and / or DENVE protein by 60%, 65%, 70%, 75%, or more. In some examples, binding is inhibited by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such competing antibodies bind to the same epitopes (eg, linear or three-dimensional epitopes) that are bound by the anti-DENV antibodies described herein. A detailed exemplary method for mapping an epitope to which an antibody binds is provided in Morris (1996) "Epitope Mapping Protocols" Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competitive assay, the immobilized DENV or DENVE E protein is associated with the first labeled antibody that binds to DENV and / or DENV E protein and the first antibody for binding to DENV or DENV E protein. Incubated in a solution containing a second unlabeled antibody tested for competing abilities. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized DENV or DENVE protein is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to DENV or DENV E protein, excess unbound antibody is removed and the amount of label bound to immobilized DENV or DENV E protein is measured. Will be done. If the amount of label bound to the immobilized DENV or DENV E protein is substantially reduced in the test sample compared to the control sample, then it is the second antibody with respect to binding to the DENV or DENVE protein. Shows that it is competing with the antibody of 1. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Assays for determining the binding activity of a polypeptide containing a mutant Fc region to one or more FcR family members are described herein or otherwise known in the art. Such binding assays include, but are not limited to, BIACORE® analytics, which include surface plasmon resonance (SPR) phenomena, amplified luminescence proximity homogenism assay (ALPHA) screening, ELISA, and fluorescence activation. Cell screening (FACS) is used (Lazar et al., Proc. Natl. Acad. Sci. USA (2006) 103 (11): 4005-4010).

In one aspect, a BIACORE® assay, eg, various FcRs as analytes, known methods and reagents (eg, protein A, protein L, protein A / G, protein G, anti-λ chain antibody, anti-. Dissociation constants (dissociation constants) obtained from analysis of sensorgrams subjected to interaction with polypeptides containing mutant Fc regions immobilized or captured on sensor chips using κ chain antibodies, antigen peptides, antigen proteins). By observing whether the Kd) value is decreasing or increasing, it is possible to determine whether the binding activity of the polypeptide containing the mutant Fc region is enhanced, maintained or decreased for a specific FcR family member. Can be used to Comparing changes in resonance unit (RU) values in sensorgrams before and after subjecting one or more types of FcR to interaction with a polypeptide containing a captured mutant Fc region as an analyte. The change in binding activity can be determined in the same manner. Alternatively, the FcR can be immobilized or captured on the sensor chip, and the polypeptide containing the mutant Fc region is used as the analyte.

In BIACORE (registered trademark) analysis, one of the substrates (ligand) in the observation of interaction is immobilized on the gold thin film on the sensor chip, and the gold thin film and glass are formed by irradiating light from the back side of the sensor chip. Due to the total internal reflection at the interface between the two, a portion of the reflected light having a reduced reflection intensity is formed (SPR signal). When the other one of the substrates (analyte) in the observation of the interaction is flushed onto the surface of the sensor chip and the ligand binds to the analyte, the mass of the immobilized ligand molecule increases and the surface of the sensor chip The refractive index of the above solvent changes. As a result of this change in index of refraction, the position of the SPR signal shifts (on the other hand, when this bond dissociates, the signal position returns). The BIACORE® system indicates the amount of shift described above, or more specifically, the time of mass by plotting the change in mass on the surface of the sensor chip as measurement data (sensorgram) on the vertical axis. Indicates a variable. The amount of analyte bound to the ligand trapped on the surface of the sensor chip is determined from the sensorgram. Dynamic parameters such as the association rate constant (ka) and the dissociation rate constant (kd) are determined from the curves of the sensorgram, and the dissociation constant (Kd) is determined from the ratio of these constants. In the BIACORE® method, a method for measuring inhibition is preferably used. Examples of methods for measuring inhibition are described in Lazar et al., Proc. Natl. Acad. Sci. USA 103 (11): 4005-4010 (2006).

ALPHA screening is performed by the ALPHA technique using two types of beads, donor and acceptor, based on the following principles. The luminescent signal is detected only when the molecule bound to the donor bead physically interacts with the molecule bound to the acceptor bead and the two beads are very close to each other. The laser-excited photosensitizer in the donor beads converts ambient oxygen into excited-state singlet oxygen. Singlet oxygen disperses around the donor beads, and when it reaches the adjacent acceptor beads, a chemiluminescent reaction is induced in the beads and finally light is emitted. If the molecule bound to the donor bead does not interact with the molecule bound to the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead and no chemiluminescent reaction occurs.

For example, the biotinylated polypeptide complex is bound to donor beads and the glutathione S transferase (GST) tagged Fc receptor is bound to acceptor beads. In the absence of a competing polypeptide complex containing a mutant Fc region, the polypeptide complex containing the parent Fc region interacts with the Fc receptor to give a signal of 520-620 nm. A polypeptide complex containing an untagged mutant Fc region competes with a polypeptide complex containing a parent Fc region for interaction with the Fc receptor. Relative binding activity can be measured by quantifying the decrease in fluorescence observed as a result of competition. Biotinlation of polypeptide complexes such as antibodies with sulfo-NHS-biotin and the like is well known. Glutathion is expressed by expressing the Fc receptor and GST in the cell carrying the fusion gene prepared by in-frame fusion of the Fc receptor-encoding polynucleotide and the GST-encoding polynucleotide in an expressible vector. The column-based purification method is suitable for adoption as a method for tagging Fc receptors with GST. The resulting signals are preferably analyzed by using software such as GRAPHPAD PRISM (GraphPad, San Diego) to fit them into a one-site competition model that uses non-linear regression analysis.

Mutant Fc regions with reduced FcR binding activity have essentially weaker binding activity than the parent Fc regions when assayed with substantially the same amount of corresponding parent Fc and mutant Fc regions. Refers to the Fc region that binds to FcR. In addition, the mutant Fc region with increased FcR binding activity binds essentially stronger than the corresponding parent Fc region when assayed with substantially the same amount of parent Fc and mutant Fc regions. It refers to the Fc region that binds to FcR with activity. The mutant Fc region that maintains FcR-binding activity is equivalent to or essentially the parent Fc region when assayed with substantially the same amount of the corresponding parent Fc region and a polypeptide containing the mutant Fc region. It refers to the Fc region that binds to FcR with the same binding activity.

Whether the binding activity of the Fc region to various FcRs has increased or decreased can be determined from the increase or decrease in the amount of binding of various FcRs to the Fc region measured according to the above method. Here, the amount of binding of various FcRs to the Fc region captures the difference in the RU value of the sensorgram that changed before and after the interaction between the various FcRs as the object to be analyzed and the Fc region, and the Fc region is captured by the sensor chip. It can be evaluated as a value divided by the difference in the RU value of the sensorgram that changed before and after. The binding activity of the Fc region to FcγR or FcRn can be measured by the method described in Example 5 herein.

In the present invention, the substantially reduced FcγR binding activity is preferably defined as, for example, that the binding activity of the mutant Fc region to FcγR is less than 50%, less than 45%, as a function of the FcγR binding activity of the parent Fc region. Less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or 0.1 Means less than%. It is also preferably, for example, [difference in RU value of sensorgram changed before and after interaction between FcγR and mutant Fc region] / [RU value of sensorgram changed before and after capturing FcγR on sensor chip]. Differences] ratio is less than 1, less than 0.8, less than 0.5, less than 0.3, less than 0.2, less than 0.1, less than 0.08, less than 0.05, less than 0.03, less than 0.02, less than 0.01, less than 0.008, less than 0.005, less than 0.003, less than 0.002 , Or less than 0.001.

In the present invention, it is preferable that the FcRn binding activity does not have substantially increased, especially at pH 7.4, preferably, for example, the binding activity of the mutant Fc region to FcRn is compared with the FcRn binding activity of the parent Fc region. Less than 1000 times, less than 500 times, less than 200 times, less than 100 times, less than 90 times, less than 80 times, less than 70 times, less than 60 times, less than 50 times, less than 40 times, less than 30 times, less than 20 times, It means less than 10 times, less than 5 times, less than 3 times, or less than 2 times. It is also preferably, for example, [difference in RU value of sensorgram changed before and after interaction between FcRn and mutant Fc region] / [RU value of sensorgram changed before and after capturing FcRn on sensor chip]. Differences] ratio is less than 0.5, less than 0.3, less than 0.2, less than 0.1, less than 0.08, less than 0.05, less than 0.03, less than 0.02, less than 0.01, less than 0.008, less than 0.005, less than 0.003, less than 0.002, or less than 0.001 Means that.

A C1q-binding ELISA can be performed to measure the binding activity of a polypeptide containing a mutant Fc region to C1q. Briefly, assay plates are coated overnight with a polypeptide containing a mutant Fc region or a polypeptide containing a parent Fc region (control) in coating buffer at 4 ° C. The plate is then washed and blocked. After washing, aliquots of human C1q are added to each well and incubated at room temperature for 2 hours. After further washing, 100 μl of sheep anti-complement C1q peroxidase-binding antibody is added to each well and incubated for 1 hour at room temperature. The plate is washed again with wash buffer and 100 μl of substrate buffer containing OPD (o-phenylenediamine dihydrochloride (Sigma)) is added to each well. The oxidation reaction observed by the yellow coloration is allowed to proceed for 30 minutes and is stopped by the addition of _{100 μl 4.5NH 2} SO _4. Then the absorbance is read at (492-405) nm. The binding activity of the Fc region to C1q can be measured by the method described in Example 4 herein.

In one aspect, the difference in C1q binding activity between the variant Fc region and the parent Fc region of the invention is less than 50%, less than 45%, less than 40%, 35% as a function of the C1q binding activity of the parent Fc region. Less than%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%.

2. Activity Assay In one aspect, an assay is provided to identify anti-DENV antibodies with biological activity. Biological activity includes, for example, blocking the binding of DENVE protein to a host cell, blocking DENV invasion into a host cell, blocking and / or preventing DENV infection in a host cell, and the like. Is done. Also provided are antibodies having such biological activity in vivo and / or in vitro.

In certain embodiments, the antibodies of the invention are tested for such biological activity. In certain embodiments, a plaque reduction neutralization test (PRNT) assay can be utilized to measure the activity or neutralization capacity of the test antibody. In some embodiments, an animal host can be used to measure anti-DENV activity in vivo.

In certain embodiments, cells can be assayed directly for binding of DENV to the test antibody. Immunohistochemical techniques, confocal techniques, and / or other techniques for assessing binding are well known to those of skill in the art. Various cell lines can be utilized in such screening assays, including cells specifically engineered for this purpose. Examples of cells used in screening assays include mammalian cells, fungal cells, bacterial cells, or viral cells. The cell can be a stimulated cell, eg, a cell stimulated with a growth factor. Those skilled in the art will appreciate that the inventions disclosed herein contemplate a wide variety of assays for measuring the ability of test antibodies to bind DENV.

In one aspect, an assay is provided to identify anti-ZIKV antibodies with biological activity. Biological activity includes, for example, blocking the binding of ZIKV to the host cell, inhibiting ZIKV invasion into the host cell, suppressing and / or preventing ZIKV infection in the host cell, and the like. Antibodies with such biological activity are also provided in vivo and / or in vitro.

In certain embodiments, the antibodies of the invention are tested for such biological activity. In certain embodiments, a plaque reduction neutralization test (PRNT) assay or a focus reduction neutralization test (FRNT) assay can be utilized to measure the activity or neutralizing potency of the test antibody. In some embodiments, an animal host can be used to measure anti-ZIKV activity in vivo.

In certain embodiments, cells can be assayed directly for binding between ZIKV and the test antibody. Immunohistochemical techniques, confocal techniques, and / or other techniques for assessing binding are well known to those of skill in the art. Various cell lines are available for such screening assays, including cells that have been genetically engineered specifically for this purpose. Examples of cells used in screening assays include mammalian cells, fungal cells, bacterial cells, or viral cells. The cell can be a stimulated cell, eg, a cell stimulated by a growth factor. Those skilled in the art will appreciate that the inventions disclosed herein are intended for a wide variety of assays for measuring the ability of test antibodies to bind ZIKV.

Depending on the assay, cell and / or tissue culture may be required. Cells can be tested using any of many different physiological assays. Alternatively, or in addition, perform molecular analysis including, but not limited to, Western blotting and / or testing of protein-protein interactions to monitor protein expression, mass spectrometry to monitor other chemical modifications, and the like. be able to.

In some embodiments, such methods utilize an animal host. For example, suitable animal hosts for the present invention can be mammalian hosts such as primates, ferrets, cats, dogs, cows, horses, and rodents such as mice, hamsters, rabbits, rats and the like. In some embodiments, the animal host is inoculated with the virus, infected with the virus, or otherwise exposed to the virus before or at the same time as the test antibody is administered. Naive animals and / or inoculated animals can be used in a variety of studies. For example, such animal models are used in virus transmission studies as is known in the art. Test antibodies can be administered to the appropriate animal host before, during, or after virus transmission studies to determine the effectiveness of the test antibody in blocking viral binding and / or infectivity to the animal host. ..

In one aspect, an assay is provided to identify a polypeptide containing a mutant Fc region with biological activity. Biological activity includes, for example, ADCC activity and CDC activity. In addition, polypeptides containing mutant Fc regions with such biological activity in vivo and / or in vitro are also provided.

In certain embodiments, polypeptides containing the mutant Fc region of the invention are tested for such biological activity. In certain aspects, the polypeptides containing the mutant Fc region of the invention modulate effector function as compared to the polypeptides containing the parent Fc region. In certain aspects, this modulation is ADCC and / or CDC modulation.

In vitro and / or in vivo cytotoxicity assays can be performed to confirm CDC activity and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody has FcγR binding (and therefore is likely to have ADCC activity) and retains FcRn binding capacity. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu Rev Immunol (1991) 9, 457-492. Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest include US Pat. No. 5,500,362 (see, eg, Hellstrom et al, Proc Natl Acad Sci USA (1986) 83, 7059-7063) and Hellstrom et al, Proc Natl Acad Sci USA (1985) 82, 1499-1502; U.S. Pat. No. 5,821,337 (see Bruggemann et al, J Exp Med (1987) 166, 1351-1361). Alternatively, non-radioactive assays can be used (eg, ACTI ™ non-radioactive cytotoxic assay for flow cytometry (Cell Technology, Mountain View, CA); and CytoTox 96® non-radioactive cells. Injury assay (see Promega, Madison, WI). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, eg, in an animal model as disclosed in Clynes et al, Proc Natl Acad Sci USA (1998) 95, 652-656. .. A C1q binding assay can also be performed to determine if the antibody has CDC activity because it binds to C1q. See, for example, the C1q and C3c binding ELISAs described in WO 2006/029879 and WO 2005/100402. CDC assays can be performed to assess complement activation (eg, Gazzano-Santoro et al, J Immunol Methods (1997) 202, 163-171; Cragg et al, Blood (2003) 101, 1045. -1052; and Cragg and Glennie, Blood (2004) 103, 2738-2743). FcRn binding and in vivo clearance / half-life measurements can also be performed using methods known in the art (see, eg, Petkova et al, Int Immunol (2006) 18, 1759-1769).

D. Immunoconjugate In some embodiments, the invention also comprises one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (eg, protein toxins, bacteria, fungi, plants or). Provided are immunoconjugates comprising the anti-DENV antibodies described herein conjugated to enzymeally active toxins of animal origin, or fragments thereof), or radioisotopes and the like. In some embodiments, the invention also comprises one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (eg, protein toxins, bacteria, fungi, plant or animal derived enzymes). Provided is an immunoconjugate comprising a polypeptide comprising the variant Fc region described herein conjugated to an active toxin, or fragment thereof), or a radioisotope or the like.

In one aspect, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including, but not limited to, such drugs: Maytansi. Noids (see US Pat. Nos. 5,208,020, 5,416,064, European Patent EP 0 425 235 B1); Auristatin, eg, monomethyl auristatin drug conjugates DE and DF (MMAE and MMAF) (US Pat. No. 5,635,483, 5,780,588). (See No. 7,498,298); Drastatin; Calicaremycin or a derivative thereof (US Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, U.S. Pat. No. 5,773,001 and No. 5,877,296; Hinman et al., Cancer Res. 53: 3336-3342 (1993); and Lode et al., Cancer Res. 58: 2925-2928 (1998)); , For example, daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al. , Bioconj. Chem. 16: 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al., J. Med. Chem. 45: 4336-4343 (2002); and US Pat. No. 6,630,579); Metotrexate; Bindesin; Taxan, eg, docetaxel, paclitaxel, larotaxel. , Tesetaxel, and Altertaxel; Tricotesen; and CC1065.

In another aspect, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, such toxins include, but are not limited to: Zifteria A. Chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modelin A chain, α-sarcin, Aleurites fordii protein, Dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, geronin, mitogerin, le Strictocin, phenomycin, enomycin, and trichothecenes.

In another aspect, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. Various radioisotopes are available for the production of radioconjugates. Examples include ^{radioactive isotopes of 211} At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb and Lu. When using a radioconjugate for detection, it is a radioactive atom for scintillography, such as Tc-99m or ¹²³ I, or nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI). Spin labels for, for example, again can include iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Compounds of antibodies to cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as: N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP). ), 4- (N-maleimidemethyl) cyclohexane-1-carboxylate succinimidyl (SMCC), iminothiolane (IT), bifunctional derivative of imide ester (eg, dimethyl HCl of adipimide acid), active ester (eg, disk of suberic acid) Synimidyl), aldehydes (eg glutaaldehyde), bis-azido compounds (eg bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg bis- (p-diazonium benzoyl) -ethylenediamine), Diisocyanates (eg, toluene 2,6-diisocyanates), and bis-active fluorine compounds (eg, 1,5-difluoro-2,4-dinitrobenzene). For example, the ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs within the cell. For example, acid-labile linkers, peptidase-sensitive linkers, light-labile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52: 127-131 (1992); US Pat. No. 5,208,020). No.) can be used.

The immunoconjugates or ADCs described herein expressly, but are not limited to, such conjugates prepared with a cross-linking agent reagent; the cross-linking agent is not limited. Not, but BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIAB , Sulfo-SMCC, and Sulfo-SMPB, as well as SVSB ((4-vinylsulfone) succinimidyl benzoate), which are commercially available (eg, Pierce Biotechnology, Rockford, IL., USA). ..

E. Methods and Compositions for Diagnosis and Detection In certain embodiments, the anti-DENV antibodies provided herein are useful for detecting the presence of DENV and / or DENVE protein in biological samples. Is. In certain embodiments, any of the anti-ZIKV antibodies provided herein are useful for detecting the presence of ZIKV in a biological sample. The term "detecting" as used herein includes quantitative or qualitative detection. In certain embodiments, the biological sample is a cell or tissue such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, sheep water, ascites. Includes plasma, primary milk, mammary gland secretions, lymph, urine, sweat, tears, gastric fluid, synovial fluid, ascites, ocular lens fluid or mucus.

In one aspect, an anti-DENV antibody for use in a diagnostic or detection method is provided. In a further aspect, a method of detecting the presence of DENV in a biological sample is provided. In certain embodiments, the method involves contacting a biological sample with an anti-DENV antibody described herein, and with the anti-DENV antibody and DENV, under conditions that allow binding of the anti-DENV antibody to DENV. Includes the step of detecting whether a complex is formed between the two. Such a method can be an in vitro method or an in vivo method. In a further aspect, a method of detecting the presence of DENVE protein in a biological sample is provided. In certain embodiments, the method involves contacting a biological sample with an anti-DENV antibody described herein, and with the anti-DENV antibody, under conditions that allow the anti-DENV antibody to bind to the DENVE protein. Includes the step of detecting whether a complex is formed with the DENVE protein. Such a method can be an in vitro method or an in vivo method. In one aspect, the anti-DENV antibody is used to select a subject eligible for treatment with the anti-DENV antibody, for example if DENV or the DENVE protein is a biomarker for patient selection.

Illustrative diseases that can be diagnosed using the antibodies of the invention include DENV infection, and diseases and / or symptoms caused by or related to DENV infection, such as dengue fever, dengue hemorrhagic fever (DHF). ), Dengue shock syndrome (DSS), etc.

In one aspect, an anti-ZIKV antibody for use in a diagnostic or detection method is provided. In a further aspect, a method of detecting the presence of ZIKV in a biological sample is provided. In certain embodiments, the method involves contacting a biological sample with an anti-ZIKV antibody described herein under conditions that allow binding of the anti-ZIKV antibody to ZIKV, and between the anti-ZIKV antibody and ZIKV. Includes the step of detecting whether or not a complex is formed in. Such a method can be an in vitro or in vivo method. In a further aspect, a method of detecting the presence of ZIKV in a biological sample is provided. In certain embodiments, the method involves contacting a biological sample with an anti-ZIKV antibody described herein under conditions that allow binding of the anti-ZIKV antibody to ZIKV, and between the anti-ZIKV antibody and ZIKV. Includes the step of detecting whether or not a complex is formed in. Such a method can be an in vitro or in vivo method. In one aspect, for example, if ZIKV is a biomarker for selecting a patient, the anti-ZIKV antibody is used to select a patient to be treated with the anti-ZIKV antibody.

Illustrative diseases that can be diagnosed using the antibodies of the invention include ZIKV infection and diseases and / or symptoms caused by or related to ZIKV infection, such as fever, rash, headache, arthralgia, red eyes and muscle. Includes pain, etc.

In certain embodiments, labeled anti-DENV antibodies are provided. In certain embodiments, labeled anti-ZIKV antibodies are provided. Labels include, but are not limited to, labels or components that are directly detected (fluorescent labels, chromophore labels, high electron density labels, chemical emission labels, radioactive labels, etc.), as well as enzymes or ligands, such as, for example. Includes components that are indirectly detected via enzymatic reactions or intermolecular interactions. Exemplary labels include, but are not limited to: radioactive isotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores such as rare earth element chelate, or fluorescein and itss. Derivatives, Rhodamine and its derivatives, Dancil, Umberiferone, Luciferase, such as firefly luciferase and bacterial luciferase (US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthin oxidase, hydrogen peroxide to oxidize dye precursors. Coupling with the enzyme used (HRP, luciferase, or microperoxidase, etc.), biotin / avidin, spin label, bacteriophage label, stable free radicals, etc.

In one aspect, the antibody containing the mutant Fc region of the present invention can be used as an affinity purifying agent. In this process, antibody variants are immobilized on a solid phase such as Sephadex resin or filter paper using methods well known in the art. The immobilized antibody variant is contacted with a sample containing the antigen to be purified, and then the support is washed with a suitable solvent, where the solvent is purified which is bound to the immobilized antibody variant. It removes substantially all substances in the sample except for the antigen to which it should be. Finally, the support is washed with another suitable solvent, such as glycine buffer pH 5.0, which releases the antigen from the antibody variant.

Antibody variants can also be useful, for example, in diagnostic assays for detecting the expression of a particular antigen in a particular cell, tissue or serum.

Antibody variants can be used in known assay methods, such as, for example, competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, (1987) pp. 147-158, CRC Press, Inc.

F. Pharmaceutical Formulations Pharmaceutical formulations of the anti-DENV antibodies described herein are such antibodies of the desired purity, one or more of any pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences). Prepared in the form of a lyophilized or aqueous solution by mixing with 16th Edition, Osol, A. (1980)). The pharmaceutical formulations of anti-ZIKV antibodies described herein are such antibodies of the desired purity, one or more of any pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th Edition, Osol, Osol, It is prepared in the form of a lyophilized preparation or an aqueous solution by mixing with A. (1980)).

Pharmaceutical formulations of a polypeptide containing the variant Fc region described herein mix such a polypeptide with the desired purity with one or more of any pharmaceutically acceptable carriers. Is prepared in the form of a lyophilized formulation or an aqueous solution.

Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used, and such carriers include, but are not limited to: Buffers such as phosphoric acid, citric acid, other organic acids; antioxidants such as ascorbic acid, methionine; preservatives (eg octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride Phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins, For example serum albumin, gelatin, immunoglobulin, etc .; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose. , Dextrin, etc .; chelating agents, such as EDTA; sugars, such as sculose, mannitol, trehalose, sorbitol, etc .; salt-forming counterions, such as sodium, etc .; metal complexes (eg, Zn-protein complexes); and / or nonionic interfaces. Activators such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as rHuPH20 (HYLENEX®). ), Baxter International) and other human soluble PH-20 hyaluronidase glycoproteins are further included. Some exemplary sHASEGPs and usages, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

An exemplary lyophilized antibody formulation is described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006/044908, the latter formulation containing histidine-acetate buffer.

The formulations described herein may also include two or more active ingredients, preferably those having complementary activities that do not adversely affect each other, if required for the particular indication to be treated. For example, but not limited to, it may be desirable to provide antiviral agents such as: interferon (eg, interferon α-2b, interferon γ, etc.), anti-DENV monoclonal antibody, anti-DENV polyclonal antibody, RNA polymerase inhibitors, protease inhibitors, helicase inhibitors, immunomodulators, antisense compounds, small interferon RNAs, small hairpin RNAs, microRNAs, RNA aptamers, ribozymes, and combinations thereof. Such active ingredients are properly present in combination in an amount effective for the intended purpose.

The active ingredient is encapsulated in, for example, microcapsules prepared by coacervation techniques or by interfacial polymerization, eg, hydroxymethyl cellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively, or colloidal drug delivery. It can be encapsulated in a system (eg, liposomes, albumin colloids, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. (1980).

It is possible to prepare a sustained release formulation. Suitable examples of sustained release formulations include a semi-permeable matrix of solid hydrophobic polymers containing antibodies, which matrix is in the form of shaped articles, such as films, or microcapsules.

The formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a disinfectant membrane.

G. Therapeutic methods and compositions Any of the anti-DENV antibodies provided herein can be used in therapeutic methods.

In one aspect, anti-DENV antibodies for use as pharmaceuticals are provided. In a further aspect, anti-DENV antibodies are provided for use in the treatment of DENV infections. In certain embodiments, anti-DENV antibodies are provided for use in therapeutic methods. In certain aspects, the invention provides an anti-DENV antibody for use in a method of treating an individual infected with DENV, the method comprising administering to the individual an effective amount of the anti-DENV antibody. In such an aspect, the method further comprises administering to the individual an effective amount, eg, at least one additional therapeutic agent, as described below. In a further aspect, the invention provides an anti-DENV antibody for use in blocking the binding of DENVE protein to a host cell and / or the invasion of DENV into the host cell. In certain aspects, the invention provides an anti-DENV antibody for use in a method that blocks the binding of DENVE protein to a host cell and / or the invasion of DENV into a host cell in an individual. The individual comprises administering to the individual an amount of anti-DENV antibody effective to block the binding of the DENVE protein to the host cell and / or the entry of DENV into the host cell. The "individual" according to any of the above embodiments is preferably human.

In a further aspect, the invention provides the use of anti-DENV antibodies in the manufacture or preparation of pharmaceuticals. In one aspect, the drug is for treating a DENV infection. In a further aspect, the drug is for use in a method of treating DENV infection, comprising the step of administering an effective amount of the drug to an individual infected with DENV. In such an aspect, the method further comprises administering to the individual an effective amount, eg, at least one additional therapeutic agent, as described below. In a further aspect, the drug is for blocking the binding of the DENVE protein to the host cell and / or the entry of DENV into the host cell. In a further aspect, the drug comprises administering to the individual an amount of drug effective to block the binding of the DENVE protein to the host cell and / or the entry of DENV into the host cell. It is intended for use in a manner that blocks the binding of DENV E protein to and / or the entry of DENV into host cells. The "individual" according to any of the above embodiments can be human.

In a further aspect, the invention provides a method for treating a DENV infection. In one aspect, the method comprises administering to an individual infected with DENV an effective amount of an anti-DENV antibody. In such an aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. The "individual" according to any of the above embodiments can be human.

In a further aspect, the invention provides a method for blocking the binding of DENVE protein to a host cell and / or the invasion of DENV into the host cell in an individual. In one aspect, the method comprises administering to the individual an amount of anti-DENV antibody effective in blocking the binding of the DENVE protein to the host cell and / or the entry of DENV into the host cell. In one aspect, the "individual" is human.

In a further aspect, the invention provides a pharmaceutical formulation comprising any of the anti-DENV antibodies provided herein, eg, for use in any of the above therapeutic methods. In one aspect, the pharmaceutical formulation comprises any of the anti-DENV antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical formulation comprises any of the anti-DENV antibodies provided herein and at least one additional therapeutic agent, eg, as described below.

In a further aspect, pharmaceutical formulations are for treating DENV infection. In a further aspect, the pharmaceutical formulation is for blocking the binding of the DENVE protein to the host cell and / or the entry of DENV into the host cell. In one aspect, the pharmaceutical product is administered to an individual infected with DENV. The "individual" according to any of the above embodiments is preferably human.

In certain embodiments, the DENV infection may include diseases and / or symptoms caused by or associated with the DENV infection, such as dengue fever, dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS).

In one aspect, the invention provides a method of treating or preventing Zika virus infection, comprising administering an antibody that binds to ZIKV. In one aspect, the invention provides a method of blocking the transmission of Zika virus from a pregnant mother to the foetation. In one aspect, the invention provides a method of preventing congenital Zika syndrome (eg, congenital developmental deficiency, microcephaly, etc.), comprising administering an antibody that binds to ZIKV. In one aspect, the invention provides a method of suppressing loss of fetal body weight (eg, whole body, head, etc.), comprising administering an antibody that binds ZIKV.

In one example, the term "congenital Zika syndrome" refers to a distinct pattern of birth defects typical of prenatally infected fetuses and newborns. As will be appreciated by those skilled in the art, subjects with congenital deca syndrome may exhibit symptoms such as, but not limited to: congenital dysgenesis, microcephaly, partial skull. Severe microcephaly that collapses, loss of brain tissue with certain patterns of brain damage such as subcortical calcification, damage to the fundus such as speckled scars and localized pigmented retinal spots, varus foot Or congenital contractions such as joint contractions, hypertonia that limits physical activity immediately after birth, etc. Administration of the antibodies described herein is believed to prevent at least one (or at least two, or at least three, or at least four, or all) symptoms of congenital deca syndrome. For example, the antibodies described herein prevent fetal or neonatal infection of prenatal Zika virus, thereby suppressing fetal weight loss (eg, systemic, head, etc.).

Any of the anti-ZIKV antibodies provided herein can be used in therapeutic methods.

In one aspect, an anti-ZIKV antibody for use as a pharmaceutical is provided. In a further aspect, anti-ZIKV antibodies are provided for use in the treatment of ZIKV infections. In certain embodiments, anti-ZIKV antibodies are provided for use in therapeutic methods. In a particular aspect, the invention provides an anti-ZIKV antibody for use in a method of treating an individual infected with ZIKV, comprising administering to the individual an effective amount of the anti-ZIKV antibody. In such an aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, eg, as described below. In a further aspect, the invention provides anti-ZIKV antibodies for use in blocking ZIKV binding and / or ZIKV invasion into host cells. In certain aspects, the invention provides an anti-ZIKV antibody for use in a method that blocks ZIKV binding and / or ZIKV invasion into a host cell in an individual, the method of which is to the individual to the host cell. It comprises administering an effective amount of anti-ZIKV antibody to block ZIKV binding and / or ZIKV invasion. The "individual" according to any of the above embodiments is preferably human.

In a further aspect, the invention provides the use of anti-ZIKV antibodies in the manufacture or preparation of pharmaceuticals. In one aspect, the drug is for treating a ZIKV infection. In a further aspect, the drug is for use in a method of treating ZIKV infection, comprising the step of administering an effective amount of the drug to an individual infected with ZIKV. In such an aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, eg, as described below. In a further aspect, the drug is for blocking ZIKV binding and / or ZIKV invasion into host cells. In a further aspect, the drug is intended to be used in a method of blocking ZIKV binding and / or invasion of a host cell in an individual, the method of which is to the individual the ZIKV binding and / or ZIKV binding to the host cell. It comprises the step of administering an effective amount of the drug to block ZIKV invasion. The "individual" according to any of the above embodiments can be human.

In a further aspect, the invention provides a method of treating a ZIKV infection. In one aspect, the method comprises administering to an individual infected with such ZIKV an effective amount of an anti-ZIKV antibody. In such an aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, eg, as described below. The "individual" according to any of the above embodiments can be human.

In a further aspect, the invention provides a method of blocking ZIKV binding and / or ZIKV invasion into a host cell in an individual. In one aspect, the method comprises administering to the individual an amount of anti-ZIKV antibody effective in blocking ZIKV binding and / or ZIKV invasion into the host cell. In one aspect, the "individual" is human.

In a further aspect, the invention provides a pharmaceutical formulation comprising any of the anti-ZIKV antibodies provided herein, eg, for use in any of the above therapeutic methods. In one aspect, the pharmaceutical formulation comprises one of the anti-ZIKV antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical formulation comprises any of the anti-ZIKV antibodies provided herein and at least one additional therapeutic agent, eg, as described below.

In a further aspect, the pharmaceutical formulation is for treating ZIKV infection. In a further aspect, the pharmaceutical formulation is for blocking ZIKV binding and / or ZIKV invasion into host cells. In one aspect, the pharmaceutical product is administered to an individual infected with ZIKV. The "individual" according to any of the above embodiments is preferably human.

In certain embodiments, ZIKV infection includes diseases and / or symptoms caused by or associated with ZIKV infection, such as fever, rash, headache, arthralgia, red eyes and myalgia.

Any of the polypeptides containing the mutant Fc region described herein can be used in therapeutic methods.

In one aspect, a polypeptide containing a mutant Fc region is provided for use as a pharmaceutical. In certain embodiments, a polypeptide comprising a mutant Fc region is provided for use in a therapeutic method. In certain aspects, the invention comprises a mutant Fc region for use in a method of treating an individual, comprising administering to the diseased individual a polypeptide comprising an effective amount of the mutant Fc region. Provide a polypeptide. In such an aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one aspect, the disease is a viral infection. In one aspect, the "individual" is human.

In a further aspect, the invention provides the use of a polypeptide containing a mutant Fc region in the manufacture or preparation of a pharmaceutical. In one aspect, the drug is for treating a disease. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In a further aspect, the drug is for use in a method of treating a disease that comprises the step of administering an effective amount of the drug to an individual having the disease to be treated. In such an aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one aspect, the disease is a viral infection. In one aspect, the "individual" is human.

In a further aspect, the invention provides a method for treating a disease. In one aspect, the method comprises administering to an individual with such a disease an effective amount of a polypeptide containing a mutant Fc region. In such an aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one aspect, the disease is a viral infection. In one aspect, the "individual" is human.

In a further aspect, the invention provides a pharmaceutical formulation containing a polypeptide comprising the mutant Fc region described herein for use in a therapeutic method, eg, any of the therapeutic methods described herein. offer. In one aspect, the pharmaceutical formulation comprises a polypeptide comprising the mutant Fc region described herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical formulation comprises a polypeptide comprising the mutant Fc region described herein and at least one additional therapeutic agent.

In a further aspect, pharmaceutical formulations are for treating the disease. In one aspect, the pharmaceutical formulation is administered to an individual with the disease. In one aspect, the disease is a viral infection. In one aspect, the "individual" is human.

The antiviral antibody containing the mutant Fc region of the present invention can suppress the antibody-dependent enhancement of infection (ADE) observed in the conventional antiviral antibody. ADE is a phenomenon in which an antibody-bound virus is phagocytosed via activated FcγR, resulting in enhanced cell infection. Fc modifications that reduce the interaction with activated FcγR may reduce the risk of ADE. Leucine to alanine mutations at positions 234 and 235 to form LALA mutants have been shown to reduce the risk of ADE infection with dengue in vivo (Cell Host Microbe (2010) 8, 271). -283). However, such modifications reduce the immune function of other antibody-mediated effectors, such as ADCC and CDC. In particular, CDC is expected to play an important role in suppressing ADE and therefore should not reduce C1q binding of the complement component of the Fc region for therapeutic effects. In addition, the half-life of the antibody can be extended by engineering manipulation of the Fc region, which alters its binding affinity to the salvage receptor FcRn, which leads to the prophylactic use of the antibody to protect against viral infections. there is a possibility.

The virus is preferably adenovirus, astrovirus, hepadonavirus, herpesvirus, papovavirus, poxvirus, arenavirus, bunyavirus, calcivirus, coronavirus, phyllovirus, flavivirus, orthomixovirus, paramixovirus, pico. It is selected from Lunavirus, Leovirus, Retrovirus, Rabdovirus, or Togavirus.

In a preferred embodiment, the adenovirus includes, but is not limited to, human adenovirus. In a preferred embodiment, the astrovirus includes, but is not limited to, mamastrovirus. In a preferred embodiment, the hepadnavirus includes, but is not limited to, hepatitis B virus. In a preferred embodiment, the herpesvirus comprises herpes simplex virus type I, herpes simplex virus type 2, human cytomegalovirus, Epstein-bar virus, varicella-zoster virus, roseolovirus, and Kaposi's sarcoma-related herpesvirus. However, it is not limited to these. In a preferred embodiment, papovavirus includes, but is not limited to, human papillomavirus and human polyomavirus. In a preferred embodiment, the pox virus is variola virus, vaccinia virus, bovine pox virus, monkey pox virus, small pox virus, pseudocow pox virus, vesicle sputitis virus, tanapox virus, yabasal tumor. Includes, but is not limited to, viruses and infectious variola virus. In a preferred embodiment, the arenavirus includes, but is not limited to, lymphocytic choriomyticitis virus, lassa virus, machupovirus, and funine virus. In a preferred embodiment, the bunyavirus includes, but is not limited to, hantavirus, nylovirus, orthobunyavirus, and frevovirus. In a preferred embodiment, the calcivirus includes, but is not limited to, vesivirus, norovirus, such as Norwalk virus, and sapovirus. In a preferred embodiment, the coronavirus includes, but is not limited to, the human coronavirus, the pathogen of Severe Acute Respiratory Syndrome (SARS). In a preferred embodiment, the filovirus includes, but is not limited to, Ebola virus and Marburg virus. In a preferred embodiment, the flavivirus is yellow fever virus, western Nile virus, dengue virus (DENV-1, DENV-2, DENV-3 and DENV-4), hepatitis C virus, tick-borne encephalitis virus, Japanese encephalitis virus, It includes, but is not limited to, Murray Valley encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Casanul forest disease virus, and Poissan encephalitis virus. In a preferred embodiment, the orthomyxovirus includes, but is not limited to, influenza A virus, influenza B virus, and influenza C virus. In a preferred embodiment, paramyxoviruses include, but are limited to, parainfluenza virus, rubravirus (Otafuku cold), morphilivirus (measles), pneumoviruses such as human respiratory syncytial virus, and subacute sclerosing panencephalitis virus. Not done. In a preferred embodiment, picornavirus includes, but is not limited to, poliovirus, rhinovirus, coxsackievirus A, coxsackievirus B, hepatitis A virus, echovirus, and enterovirus. In a preferred embodiment, the reovir includes, but is not limited to, the Colorado tick fever virus and rotavirus. In a preferred embodiment, retroviruses include, but are not limited to, lentiviruses such as human immunodeficiency virus and human T-lymphotropic virus (HTLV). In a preferred embodiment, rhabdoviruses include, but are not limited to, lyssaviruses such as rabies virus, vesicular stomatitis virus, and infectious hematopoietic necrosis virus. In a preferred embodiment, the togavirus includes alpha viruses such as Ross River virus, Onyonnyon virus, Sindbis virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, and Western equine encephalitis virus, as well as eczema virus. Not limited.

In a further aspect, the invention provides, for example, a method of preparing a pharmaceutical or pharmaceutical formulation for use in any of the above therapeutic methods, wherein the method is any of the anti-DENV antibodies described herein. Includes the step of mixing the antibody with a pharmaceutically acceptable carrier. In one aspect, the method of preparing a pharmaceutical or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the pharmaceutical or pharmaceutical formulation.

In a further aspect, the invention provides a method of preparing a pharmaceutical or pharmaceutical formulation for use, for example, in any of the above therapeutic methods, the method of which is the anti-ZIKV antibody provided herein. It comprises the step of mixing either with a pharmaceutically acceptable carrier. In one aspect, the method of preparing a pharmaceutical or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the pharmaceutical or pharmaceutical formulation.

The antibodies of the invention can be used alone or in combination with other agents in therapeutic methods. For example, the antibodies of the invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an antiviral agent, eg, but not limited to, interferon (eg, interferon α-2b, interferon γ, etc.), anti-DENV monoclonal antibody, anti-DENV polyclonal antibody, RNA polymerase. Inhibitors, protease inhibitors, helicase inhibitors, immunomodulators, antisense compounds, small interferon RNAs, small hairpin RNAs, microRNAs, RNA aptamers, ribozymes, and combinations thereof.

In a further aspect, the invention provides a method of preparing a pharmaceutical or pharmaceutical formulation for use, for example, in any of the above therapeutic methods, the method comprising the mutant Fc region described herein. It comprises the step of mixing any of the comprising polypeptides with a pharmaceutically acceptable carrier. In one aspect, the method of preparing a pharmaceutical or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the pharmaceutical or pharmaceutical formulation.

Polypeptides containing the mutant Fc region of the present invention can be used alone or in combination with other agents in therapeutic methods. For example, a polypeptide containing the mutant Fc region of the invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an antiviral agent, eg, but not limited to, interferon (eg, interferon α-2b, interferon γ, etc.), antiviral monoclonal antibody, antiviral polyclonal antibody, RNA polymerase. Inhibitors, protease inhibitors, helicase inhibitors, immunomodulators, antisense compounds, small interferon RNAs, small hairpin RNAs, microRNAs, RNA aptamers, ribozymes, and combinations thereof.

Such combination therapies described above include combination administration (when two or more therapeutic agents are contained in the same formulation or separate formulations), and individual administration, and in the case of individual administration, the antibody of the present invention or The administration of the polypeptide containing the mutant Fc region can be performed before, at the same time as, and / or following the administration of the additional therapeutic agent (s). In one aspect, administration of the anti-DENV antibody and administration of additional therapeutic agents to each other within about 1 month, within about 1, 2 or 3 weeks, or within about 1, 2, 3, 4, 5 or 6 days. Will be done. In another aspect, administration of the polypeptide containing the mutant Fc region and administration of additional therapeutic agents to each other within about 1 month, about 1, 2 or 3 weeks, or about 1, 2, 3, 4, It takes place within 5 or 6 days.

Polypeptides (and any additional therapeutic agents) containing the antibodies or mutant Fc regions of the invention are desired for topical treatment by any suitable means, eg, parenteral, intrapulmonary, and intranasal administration. If so, it can be administered by intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be done in part by any suitable route, depending on whether the administration is short-term or long-term, for example by injection, such as intravenous or subcutaneous injection. Different dosing schedules, such as single or multiple doses at different time points, bolus doses, pulse injections, etc., are contemplated herein.

The antibody or polypeptide containing the mutant Fc region of the present invention will be formulated and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disease to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the drug, the method of administration, the dosing regimen, and Other factors known to healthcare professionals are included. This agent, but not necessarily, may optionally be formulated with one or more agents currently used to prevent or treat the disease in question. The effective amount of such other drug depends on the amount of drug present in the formulation, the type of disease or treatment, and the other factors mentioned above. These are generally determined empirically / clinically to be appropriate, at the same doses and routes as those described herein, or at approximately 1-99% of the doses described herein. It is used in any dose and by any route.

When preventing or treating a disease, the appropriate dose of the antibody or polypeptide containing the variant Fc region of the invention (when used alone or in combination with one or more other therapeutic agents) is , Depends on the following factors: type of disease to be treated, type of antibody, type of polypeptide containing mutant Fc region, severity and course of disease, polypeptide containing antibody or mutant Fc region for prophylactic purposes Whether administered in or for therapeutic purposes, previous treatment, patient history and patient response to antibodies or polypeptides containing the variant Fc region, and the judgment of the attending physician. The antibody or polypeptide containing the mutant Fc region is administered to the patient at once or appropriately over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (eg, 0.1 mg / kg to 10 mg / kg) of the antibody or polypeptide containing the variant Fc region, eg, one or more separate It can be the initial candidate dose for administration to a patient, whether by administration or by continuous infusion. A typical daily dose can range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated doses over several days or longer, depending on the disease, treatment is generally continued until the desired suppression of disease symptoms occurs. One exemplary dose of an antibody or polypeptide containing a mutant Fc region ranges from about 0.05 mg / kg to about 10 mg / kg. Thus, about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient one or more times. Such doses are intermittent (eg, such that the patient receives the antibody or polypeptide containing the mutant Fc region about 2 to about 20 times, eg about 6 times), eg once a week or once every 3 weeks. Can be administered once. Initially higher loading doses can be administered, followed by one or more lower doses. The progress of this treatment is easily monitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeutic methods can be performed in place of or in addition to anti-DENV or anti-ZIKV antibodies using the immunoconjugates of the invention. Similarly, it is understood that any of the above formulations or therapeutic methods can be performed using the immunoconjugates of the invention in place of or in addition to the polypeptides containing the mutant Fc regions described herein. NS.

H. Manufactured Product In another aspect of the present invention, a manufactured product containing a substance / material useful for the treatment, prevention and / or diagnosis of the above-mentioned diseases is provided. The product includes the container and the label on the container or the package insert enclosed with the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion bags and the like. The container can be made of various materials such as glass or plastic. The container may contain the composition alone or in combination with other compositions effective in treating, preventing and / or diagnosing the disease and may have a sterile access port (eg,). , The container can be an intravenous infusion bag or a vial with a stopper that can be punctured by a hypopodermic needle). At least one active ingredient in the composition is an antibody of the invention or a polypeptide containing a mutant Fc region. The label or package insert indicates that the composition will be used in the treatment of a given disease. In addition, the product contains (a) a first container containing a composition containing a polypeptide containing an antibody or mutant Fc region of the invention; and (b) an additional cytotoxic agent or other therapeutic agent. A second container containing the composition to be contained can be included. The product of this aspect of the invention may further include a package insert indicating that the composition can be used in the treatment of a particular disease. Alternatively, or in addition, the product contains a second (or third) pharmaceutically acceptable buffer, such as bacteriostatic injection water (BWFI), phosphate buffered saline, Ringer's solution, dextrose solution, and the like. Additional containers may be included. It can further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, and the like.

It is understood that any of the above products may contain the immunoconjugates of the invention in place of or in addition to anti-DENV or anti-ZIKV antibodies. Similarly, it is understood that any of the above products may contain the immunoconjugates of the invention in place of or in addition to the polypeptide containing the mutant Fc region.

III. Examples The following are examples of the methods and compositions of the present invention. It will be appreciated that various other aspects can be implemented, taking into account the general description above.

Example 1: Preparation of antigen and antibody
Expression and Purification of Recombinant Soluble E Proteins Derived from DENV-1, DENV-2, DENV-3, and DENV-4 DENV-1, DENV-2, DENV-3, and DENV with 8 x histidine tag at the carboxy terminus Recombinant soluble E protein (0.8E-His) derived from -4 (SEQ ID NO: 65-68, respectively) using FreeStyle293-F cell line or Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). It was expressed transiently. By expressing prM0.8E-His (SEQ ID NO: 61 to 64, respectively) derived from DENV-1, DENV-2, DENV-3, and DENV-4, prM0.8E-His is simply expressed in the cells. Expressed as a single polypeptide; this polypeptide is then intracellularly processed and cleaved between prM and 0.8E-His. As a result, 0.8E-His was secreted into the cell culture medium. The conditioned medium containing 0.8E-His was applied to a column packed with an immobilized metal affinity chromatography (IMAC) resin supplemented with nickel or cobalt, followed by elution with imidazole. Fractions containing 0.8E-His were pooled and applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing 0.8E-His were pooled and stored at -80 ° C.

Expression and purification of recombinant human FcγR The extracellular domain of human FcγR was prepared by the following method. First, the gene for the extracellular domain of FcγR was synthesized by a method well known to those skilled in the art. At that time, the sequence of each FcγR was prepared based on the information registered in NCBI. Specifically, FcγRIa was prepared based on the sequence of NCBI accession number NM_000566 (version number NM_000566.3), FcγRIIa was prepared based on the sequence of NCBI accession number NM_001136219 (version number NM_001136219.1), and FcγRIIb was prepared. Was made based on the sequence of NCBI accession number NM_004001 (version number NM_004001.3), FcγRIIIa was made based on the sequence of NCBI accession number NM_001127593 (version number NM_001127593.1), and FcγRIIIb was made based on the sequence of NCBI accession number NM_000570. It was made based on the sequence of (version number NM_000570.3) and His tag was attached to the C-terminal of each FcγR construct. In addition, the presence of polymorphisms is known for FcγRIIa, FcγRIIIa, and FcγRIIIb, Warmerdam et al. (J Exp Med (1990) 172, 19-25) for FcγRIIa and Wu et al. For FcγRIIIa. Polymorphic sites were prepared by referring to (J Clin Invest (1997) 100, 1059-1070) and Ory et al. (J Clin Invest (1989) 84, 1688-1691) for FcγRIIIb.

An expression vector was constructed by inserting the obtained gene fragment into an animal cell expression vector. The constructed expression vector was transiently introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cancer cells to express the target protein. The liquid prepared by filtering the culture supernatant obtained from the transiently introduced cell culture solution with a 0.22 μm filter was, in principle, purified by the following four steps: (i) cation exchange column chromatography. Graphics (SP Sepharose FF); (ii) Affinity column chromatography for His tags (HisTrap HP); (iii) Gel filtration column chromatography (Superdex 200); and (iv) Sterilization filtration. Anion exchange column chromatography with Q Sepharose FF was used in step (i) to purify FcγRI. The absorbance of the purified protein was measured at 280 nm using a spectrophotometer. Based on the measured values, the concentration of purified protein was calculated using the absorption coefficient determined by a method such as PACE (Protein Science (1995) 4, 2411-2423).

Expression of recombinant mouse FcγR and purification The extracellular domain of mouse FcγR (mFcγR) was prepared by the following method. First, the gene for the extracellular domain of FcγR was synthesized by a method generally known to those skilled in the art. In this synthesis, sequences of each FcγR were prepared based on the information registered in NCBI. Specifically, mFcγRI was prepared based on the sequence of NCBI reference sequence: NP_034316.1, mFcγRIIb was prepared based on the sequence of NCBI reference sequence: NP_034317.1, and mFcγRIII was prepared based on the sequence of NCBI reference sequence: NP_034318.2. And mFcγRIV was made based on the sequence of NCBI reference sequence: NP_653142.2. A His tag was added to each of these sequences at the C-terminus.

Each of the obtained gene fragments was inserted into a vector for expression in animal cells to prepare an expression vector. The prepared expression vector was transiently introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cancer cells to express the target protein. After collecting the obtained culture supernatant, the culture supernatant was obtained by passing through a 0.22 μm filter. In principle, the obtained culture supernatant was purified by the following four steps: (i) ion exchange column chromatography; (ii) affinity column chromatography for His tags (HisTrap HP); (iii) gel filtration column. Chromatography (Superdex 200); and (iv) Sterilization filtration. Ion exchange column chromatography in step (i) was performed using Q Sepharose HP for mFcγRI, SP Sepharose FF for mFcγRIIb and mFcγRIV, and SP Sepharose HP for mFcγRIII. In the subsequent steps (iii), D-PBS (-) was used as the solvent, but D-PBS (-) containing 0.1 M arginine was used for mFcγRIII. The absorbance of each purified protein was measured at 280 nm using a spectrophotometer, and the purified protein was used from the obtained value using the extinction coefficient calculated by a method such as PACE (Protein Science (1995) 4, 2411-2423). The concentration of was calculated.

Expression and purification of recombinant human FcRn
FcRn is a heterodimer of FcRnα chain and β2-microglobulin. Oligo DNA primers were prepared based on the published human FcRn gene sequence (J Exp Med (1994) 180, 2377-2381). Using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the prepared primers, DNA fragments encoding all genes were prepared by PCR. Using the obtained DNA fragment as a template, a DNA fragment encoding an extracellular domain (Met1-Leu290) containing a signal region was amplified by PCR and inserted into a mammalian cell expression vector. Similarly, oligo DNA primers were prepared based on the published human β2-microglobulin gene sequence (Proc Natl Acad Sci USA (2002) 99, 16899-16903). Using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the prepared primers, DNA fragments encoding all genes were prepared by PCR. Using the obtained DNA fragment as a template, a DNA fragment encoding all proteins including the signal region (Met1-Met119) was amplified by PCR and inserted into a mammalian cell expression vector.

Soluble human FcRn was expressed by the following procedure. A plasmid constructed for the expression of human FcRnα chain (SEQ ID NO: 81) and β2-microglobulin (SEQ ID NO: 82) was applied to cells of the human embryonic kidney cancer-derived cell line HEK293H (Invitrogen) into PEI. It was introduced by the lipofectamine method using (Polyscience). The obtained culture supernatant was collected, and FcRn was purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences) and further purified using HiTrap Q HP (GE Healthcare) (J Immunol (2002). 169, 5171-5180).

Expression and purification of recombinant antibody
Recombinant antibodies were transiently expressed using either the FreeStyle293-F cell line or the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Purification from the acclimatized culture medium expressing the antibody was carried out by a conventional method using protein A. Further gel filtration was performed as needed.

Expression and purification of recombinant soluble human CD154 The human CD154 gene was synthesized based on the published protein sequence (NP_000065.1). A DNA fragment encoding a soluble form of human CD154 (shCD154) with a FLAG tag was prepared by PCR using the synthesized DNA as a template, and the obtained DNA fragment was inserted into a mammalian cell expression vector, thereby FLAG. -ShCD154 (SEQ ID NO: 106) expression vector was prepared. The nucleotide sequence of the resulting expression vector was determined using conventional methodologies known to those of skill in the art. FLAG-shCD154 was expressed using a FreeStyle 293 cell line (Invitrogen) as described by the protocol provided by the manufacturer. After transfection, the cells were grown for an appropriate period of time before the conditioned culture was collected. The conditioned culture was subjected to cation exchange chromatography using 25 mM MES (pH 6.0) to elute FLAG-shCD154 with a continuous gradient of 25 mM MES, 1 M NaCl (pH 6.0). Peak fractions were pooled, concentrated using Amicon Ultra Ultracel and subjected to gel filtration chromatography using phosphate buffered saline (Wako). The peak fractions were pooled again, concentrated using Amicon Ultra Ultracel, and then filtered through a 0.22 μm PVDF membrane filter for sterilization. Absorbance was measured at 280 nm using a spectrophotometer to determine the concentration of purified FLAG-shCD154. From the measured values, the protein concentration was calculated using the absorption coefficient calculated by the method described in Protein Science (1995) 4: 2411-2423.

Example 2: Preparation of antibody mutant with improved affinity for DENV E protein Genes encoding VH (3CH, SEQ ID NO: 1) and VL (3CL, SEQ ID NO: 7) of anti-DENV E protein antibody Synthesized and combined with human IgG1 CH (SG182, SEQ ID NO: 46) and human CL (SK1, SEQ ID NO: 60), respectively, both constructs were cloned into a single expression vector. This antibody is referred to herein as DG_3CH-SG182 / 3CL-SK1 or 3C.

Many mutations and their combinations were examined to identify mutations and their combinations that improve the binding properties of 3C. Next, multiple mutations were introduced into the variable region to increase the binding affinity for the E protein. Thus optimized VH mutants 3CH912 (SEQ ID NIO: 2), 3CH953 (SEQ ID NO: 3), 3CH954 (SEQ ID NO: 4), 3CH955 (SEQ ID NO: 5), 3CH1047 (SEQ) ID NO: 6), 3CH987 (SEQ ID NO: 90), 3CH989 (SEQ ID NO: 91), 3CH992 (SEQ ID NO: 92), 3CH1000 (SEQ ID NO: 93), 3CH1046 (SEQ ID NO: 94) , 3CH1049 (SEQ ID NO: 95), and the optimized VL variants 3CL499 (SEQ ID NO: 8), 3CL563 (SEQ ID NO: 9), 3CL658 (SEQ ID NO: 10), 3CL012 (SEQ ID NO: 10), 3CL012 (SEQ ID NO: 10) ID NO: 96), 3CL119 (SEQ ID NO: 97), 3CL633 (SEQ ID NO: 98), 3CL666 (SEQ ID NO: 99), 3CL668 (SEQ ID NO: 100) were obtained. A gene encoding VH is combined with human IgG1 CH (SG182, SEQ ID NO: 46; SG1095, SEQ ID NO: 54; or SG1106, SEQ ID NO: 59), and a gene encoding VL is used. Combined with human CL (SK1, SEQ ID NO: 60). Each of them was cloned into an expression vector. The amino acid sequences of the antibody variants are shown in Table 2. One of the variants, DG_3CH1047-SG182 / 3CL658-SK1, is also referred to herein as 3Cam, and the other variant, DG_3CH1047-SG182 / 3CL-SK1, is referred to herein as 3Cam2.

The antibody was expressed in HEK293 cells cotransfected with a mixture of heavy chain expression vector and light chain expression vector and purified with protein A.

(Table 2) Amino acid sequences of 3C and 3C mutants

The affinity of anti-DENV E protein antibodies that bind to the E proteins of DENV-1, DENV-2, DENV-3, and DENV-4 was measured at 25 ° C. using a Biacore T200 instrument (GE Healthcare). An anti-histidine antibody (GE Healthcare) was immobilized on all flow cells of the CM4 sensor chip using an amine coupling kit (GE Healthcare). All antibodies and analytes were prepared in PBS at pH 7.4 containing 20 mM sodium phosphate, 150 mM NaCl, 0.05% Tween 20 and 0.005% NaN _3. The E proteins of DENV-1, DENV-2, DENV-3, and DENV-4 with C-terminal His tags were captured in flow cell 2 or 3, and flow cell 1 was used as the reference flow cell. The E-protein capture level was aimed at 200 resonance units (RU). Anti-DENVE protein antibody was injected over the sensor surface at 250 nM for 180 seconds, followed by dissociation for 300 seconds. After each cycle, the sensor surface was regenerated with 10 mM Gly-HCl pH 1.5. Binding affinity was measured by processing the data using Biacore T200 analysis software, version 2.0 (GE Healthcare) and fitting it to a 1: 1 binding model.

Tables 3a and 3b show DENV-1 (denoted as DV1 in the table), DENV-2 (denoted as DV2 in the table), DENV-3 (denoted as DV3 in the table), and DENV-4 (denoted as DV4 in the table). _{The affinity (K d} ) of the anti-DENV E protein antibody that binds to the E protein of. Each 3C mutant showed increased binding affinity for all four DENV serotypes compared to parental 3C. As an example, the sensorgrams of the parent antibody 3C (DG_3CH-SG182 / 3CL-SK1) and one mutant antibody 3Cam (DG_3CH1047-SG182 / 3CL658-SK1) are shown in FIG. In addition, the sensorgrams of the parent antibody 3C (DG_3CH-SG182 / 3CL-SK1) and one mutant antibody 3Cam2 (DG_3CH1047-SG182 / 3CL-SK1) are shown in FIG.

注：* 強い結合物質、遅いオフ速度＜1E-05、KDは一意に決定され得ない。 (Table 3a) Kd values of 3C and 3C mutants for 4 DENV serotypes

Note: * Strong binding material, slow off-speed <1E-05, KD cannot be uniquely determined.

注：* 強い結合物質、遅いオフ速度＜1E-05、KDは一意に決定され得ない。 (Table 3b) Kd values of 3C and 3C mutants for DENV1 and DENV3 serotypes

Note: * Strong binding material, slow off-speed <1E-05, KD cannot be uniquely determined.

Example 3: Preparation of antibody CH mutants for improved properties Multiple mutations were introduced into human IgG1 heavy chain constant region CH (SG182, SEQ ID NO: 46). As a result, human IgG1 CH mutants SG192 (SEQ ID NO: 47), SG1085 (SEQ ID NO: 48), SG1086 (SEQ ID NO: 49), SG1087 (SEQ ID NO: 50), SG1088 (SEQ ID) NO: 51), SG1089 (SEQ ID NO: 52), SG1090 (SEQ ID NO: 53), SG1095 (SEQ ID NO: 54), SG1096 (SEQ ID NO: 55), SG1097 (SEQ ID NO: 56), SG1098 (SEQ ID NO: 57), SG1105 (SEQ ID NO: 58), SG1106 (SEQ ID NO: 59), SG1109 (SEQ ID NO: 107), SG1044 (SEQ ID NO: 108), and SG1045 (SEQ ID) NO: 109) was prepared. The gene encoding the CH mutant is the VH of 3C (3CH, SEQ ID NO: 1), the VH of one 3C mutant (3CH1047, SEQ ID NO: 6), or the VH of the anti-CD154 antibody (SLAPH0336a, SEQ ID NO: 6). : 88) combined. The VL gene (SLAPL0336a, SEQ ID NO: 89) of the anti-CD154 antibody was combined with human CL (SK1, SEQ ID NO: 60). Each of them was cloned into an expression vector. Details of the CH mutants are summarized in Table 4. To assess the avidity binding of CH mutants to each Fc receptor, the variable region SLAPH0336a / SLAPL0366a of the anti-CD154 antibody, which is capable of forming large immune complexes in the presence of the trimer antigen CD154, was used.

The antibody was expressed in HEK293 cells cotransfected with a mixture of heavy chain expression vector and light chain expression vector and purified with protein A.

(Table 4) Amino acid sequence of mutant Fc region

Example 4: Binding affinity of antibody with Fc mutant to complement C1q
Human C1q binding assay
An anti-CD154 antibody having an Fc mutant (an in-house antibody prepared by the method described in Example 3) was dispensed into Nunc-Immuno Plate MaxiSorp (Nalge Nunc International) at 4 ° C. It was left to stand at night. After washing with PBST, the plate was blocked for 2 hours at room temperature with TBST containing 0.5% BSA and 1 × Block Ace: Blocking Agent (DS Pharma). After washing the plate, human C1q (Calbiochem) was dispensed into the plate and allowed to stand at room temperature for 1 hour. The plate was washed, HRP-labeled anti-human C1q antibody (Bio Rad) was added, and the mixture was reacted at room temperature for 1 hour for washing. Subsequently, a TMB substrate (Invitrogen) was added. The signal was measured with a plate reader at wavelengths of 450 nm (test wavelength) and 570 nm (reference wavelength). The binding affinity of an antibody (WT) having a wild-type Fc region to human C1q is reduced by introducing a LALA mutation into the Fc region, and the reduction of binding affinity to human C1q is reduced by introducing KWES, EFT in addition to the LALA mutation. , Or by further introducing EFT + AE (Fig. 2). The LALA + KMES mutation slightly increased the binding affinity, while the LALA + KWES, LALA + KAES, and LALA + KEES mutants bound to C1q with an affinity comparable to WT (Fig. 3). The binding properties of the above LALA or LALA + KAES mutants were unaffected by further introduction of the ACT3 or ACT5 mutation (Fig. 4).

Mouse C1q binding assay
An anti-CD154 antibody having an Fc mutant (an in-house antibody prepared by the method described in Example 3) was dispensed into Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 ° C. After washing with PBST, the plate was blocked at 4 ° C. for 7 hours with TBST containing 0.5% BSA and 1 × Block Ace: Blocking Agent (DS Pharma). After washing the plate, 10% mouse plasma (Innovative Research) was dispensed into the plate and allowed to stand overnight at 4 ° C. The plate was washed, biotinylated anti-mouse C1q antibody (Hycult Biotech) was added, and the mixture was reacted at room temperature for 1 hour for washing. Streptavidin-HRP (Pierce) was added, reacted at room temperature for 1 hour, and washed. Subsequently, ABTS ELISA HRP substrate (KPL) was added. The signal was measured with a plate reader at a wavelength of 405 nm. The binding affinity of an antibody (WT) with a wild-type Fc region to mouse C1q was reduced by introducing a LALA mutation into the Fc region, and the reduced binding affinity to mouse C1q further reduced KAES in addition to the LALA mutation. It recovered by introducing it. The binding properties of the above LALA or LALA + KAES mutants were unaffected by further introduction of the ACT3 or ACT5 mutation (Fig. 5).

Example 5: Biacore analysis of Fc mutants that bind to FcγR and FcRn
Binding of Fc variants to human or mouse FcγR and human FcRn at pH 7.4 was measured at 25 ° C. using a Biacore T200 instrument (GE Healthcare). All antibodies and FcγR or FcRn were prepared in PBS-P at pH 7.4 containing 50 mM Na phosphate, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN _3. In the FcγR binding assay, an anti-histidine antibody (GE Healthcare) was immobilized on all flow cells of the CM4 sensor chip using an amine coupling kit (GE Healthcare). Each of the FcγRs was captured in flow cells 2, 3, or 4 with an anti-histidine antibody and flow cell 1 was designated as a reference flow cell. The FcγR capture level was aimed at 400 resonance units (RU). All antibodies were injected into all flow cells at 100 nM. Immune complexes were prepared by mixing the antibody and trimer CD154 in a 1: 1 molar ratio and incubated for 1 hour at room temperature. The sensor surface was regenerated with 10 mM glycine-HCl pH 1.5 after each cycle.

In the FcRn binding assay, the Biotin CAPture kit (GE Healthcare) was used to immobilize the Biotin CAPture reagent (GE Healthcare) on both flow cells 1 and 2 of the CAP sensor chip. Biotinylated FcRn was captured in flow cell 2 with flow cell 1 as a reference flow cell. The FcRn capture level was aimed at 400 RU. All antibodies were injected into flow cells 1 and 2 at 100 nM. Immune complexes were prepared by mixing the antibody and trimer CD154 in a 1: 1 molar ratio and incubated for 1 hour at room temperature. The sensor surface was regenerated with 8M guanidine-HCl, 1M NaOH (3: 1 vol / vol) after each cycle.

Binding levels were normalized to the corresponding FcγR or FcRn capture levels. Binding of the antibody alone or immune complex (antibody and trimer CD154 antigen) to human or mouse FcγR and human FcRn was monitored based on the binding response. Immune complexes were used to assess enhanced binding to FcγR or FcRn by the avidity effect. The results for humans FcγR1a, FcγR2a 167H and 167R, FcγR2b, FcγR3a 158F and 158V, FcγR3b NA1 and NA2 are shown in FIGS. 6 (a) to 6 (h). The results for mice FcγR1, FcγR2b, FcγR3, and FcγR4 are shown in FIGS. 7 (a) to 7 (d). Binding of the wild-type Fc region (denoted as WT IgG) was significantly reduced by introducing LALA, LALA + KAES or LALA + KWES mutations into the Fc region for each of the FcγRs tested. The tendency was similar between assays using antibody alone (described as Ab alone) and immune complex (described as CD154 IC). Binding of KAES or KWES mutants was not significantly reduced for most of the FcγRs tested compared to WT IgG binding. The results for human FcRn are shown in FIG. Binding of the wild-type Fc region (denoted as hIgG1) to human FcRn appeared to be unaffected even after introducing the LALA or LALA + KAES mutation into the Fc region. The binding was slightly enhanced by further introduction of the ACT3 or ACT5 mutation, but remained relatively low (Fig. 8).

Example 6: In vivo efficacy of anti-DENV antibody against DENV infection
6-8 week old AG129 mice ^{were intraperitoneally infected with 10 6} plaque forming units (pfu) DENV-2 strain D2Y98P. After 48 hours, mice were treated with 1-30 μg antibody in P1 buffer. The antibody was injected intravenously through the posterior orbital pathway. Blood was collected 24 hours later, 72 hours after the initial infection. In a previous study (Zust et al, J Virol (2014) 88, 7276-7285; Tan et al, PLOS Negl Trop Dis (2010) 4, e672), the peak of viremia after D2Y98P infection was 3 after infection. It has been proven to reach in ~ 4 days. Viral RNA was extracted from plasma from each mouse and quantitative PCR was performed to compare it with the DENV-2 standard, which has known infectivity in the plaque assay. Both 3C and 3Cam antibodies significantly reduced viremia in this mouse model compared to PBS controls, and both antibodies were comparable in efficacy. Antibodies with the LALA + KAES mutation in the Fc region showed stronger efficacy than antibodies with only the LALA mutation. This was true for both 3C and 3Cam antibodies (Figure 9). This result indicates that the restoration of C1q binding activity by adding the KAES mutation to the LALA mutation contributes to the antiviral effect of the antibody.

DENV virus (DENV-1 strain 08K3126, DENV-2 strain D2Y98P, DENV-3 strain VN32 / 96, DENV-4 strain TVP360) of ^{10 6} to 10 ⁷ plaque forming units (pfu) in 6 to 8 week old AG129 mice ) Was intraperitoneally infected. After 48 hours, mice were treated with 1-30 μg antibody in P1 buffer. The antibody was injected intravenously through the posterior orbital pathway. Blood was collected 24 hours later, 72 hours after the initial infection. In a previous study (Zust et al, J Virol (2014) 88, 7276-7285; Tan et al, PLOS Negl Trop Dis (2010) 4, e672), the peak of viremia after D2Y98P infection was 3 after infection. It has been proven to reach in ~ 4 days. Viral RNA was extracted from plasma from each mouse and quantitative PCR was performed to compare it to the DENV standard with known infectivity in the focus formation assay. Both 3C and 3Cam2 antibodies with the same Fc variant (LALA + KAES) significantly reduced viremia compared to P1 buffer control in this mouse model, and 3Cam2 antibody was viral blood compared to 3C antibody. The disease was alleviated (Fig. 10).

3Cam2 with the LALA + KAES mutation in the Fc region showed stronger efficacy as the antibody dose was increased compared to the antibody with only the LALA mutation (Fig. 11). This result indicates that the restoration of C1q binding activity by adding the KAES mutation to the LALA mutation contributes to the antiviral effect of the antibody.

Although the above invention has been described in some detail by description and examples for the purpose of clarifying understanding, the description and examples should not be construed as limiting the scope of the present invention. All patent and scientific literature disclosures cited herein are expressly incorporated herein by reference in their entirety.

Example 7: In vitro test of cross-reactivity of dengue-specific antibodies 3C and 3Cam2 to Zika virus
7.1. FRNT Assay A focus-reducing neutralization (FRNT) assay was performed to test the neutralization capacity of DG_3CH1047-SG1106 / 3CL_SK1 (or 3Cam2-LALA + KAES + ACT5) against deer. The results revealed that 3Cam2-LALA + KAES + ACT5 has a higher neutralizing ability against deer than DG_3CH-SG1106 / 3CL_SK1 (or 3C-LALA + KAES + ACT5) (Fig. 13). The average neutralizing capacity or EC50 against Zika virus is provided in Table 5.

The sequence of the antibody tested is as follows.

(Table 5) Neutralizing ability of antibody 3Cam2-LALA + KAES + ACT5 and 3C-LALA + KAES + ACT5 against deer

7.2. ADE assay with Zika virus
To test whether 3Cam2-LALA + KAES + ACT5 suppresses not only enhancement of DENV infection but also enhancement of Zika virus infection compared to the wild-type Fc version of the antibody, an ADE assay using K562 cells was performed. I went (Fig. 15). The methodology is the same as that used for DENV. Enhancement was observed in the wild-type Fc version of DG_3CH1047-SG182 / 3CL_SK1 (or 3Cam2), but not in its Fc mutants DG_3CH1047-SG1095 / 3CL_SK1 (or 3Cam2-LALA + KAES) and 3Cam2-LALA + KAES + ACT5 (Fig. 15A). .. This experiment was repeated with a new batch of 3Cam2 Ab to confirm the suppression of infection enhancement by the Fc mutant antibody. Overall, the data show that LALA mutations suppress the enhancement of both DENV and Zika virus infections, and that additional KAES and ACT5 mutations do not reduce the effects of LALA.

7.3. In vitro method
7.3.1. Virus Zika virus (Asian strain, accession number KF993678) was obtained from the Environmental Health Institute in Singapore. The virus was grown in C6 / 36 cells (ATCC) and quantified using a focus formation assay as follows: 20-24 hours before infection, 1 x 105 BHK-21 cells per well. Seeded in 24-well plates and incubated overnight at 370C, 5% CO2. The virus-containing supernatant was serially diluted 1: 100 to 1: 100,000 and incubated at 370 C, 5% CO2 for 2-3 hours in a volume of 500 μL above the cell monolayer. At the end of the incubation, each well was covered with 0.5 mL of 0.8% methylcellulose in RPMI. The plates were incubated at 370C, 5% CO2 for 4 and a half days.

For staining of infected cells, cell culture medium containing methylcellulose was removed and cells were fixed with 3.7% formaldehyde for 30 minutes. After removal of formaldehyde, the plates were washed with tap water and permeabilized with 1% Triton X-100 in 1X PBS. After a further wash step, infected cells were stained with antibody 4G2 diluted in 1% FBS / 1X PBS and then incubated with polyclonal goat anti-mouse HRP diluted 1: 2,000 in 1% FBS / PBS. A Trueblue peroxidase substrate (KPL) was used for the color reaction. Focus was manually counted for each well and the initial concentration was determined based on the average of the numbers from all countable wells.

7.3.2. FRNT Assay 20-24 hours prior to infection, 1 x 105 BHK-21 cells per well were seeded in 24-well plates and incubated overnight at 370C, 5% CO2. The antibody diluent was prepared in RPMI medium on a 96U bottom sterile plate and a certain amount of virus was added. The mixture was incubated at 37oC, 5% CO2 for 30 minutes before being added to the cells. Overnight culture medium from a 24-well plate was replaced with 450 μL of fresh 5% FBS / RPMI, 50 μL of virus / antibody mixture was added to the wells and incubated at 370 C, 5% CO2 for 2-3 hours.

At the end of the incubation, each well was covered with 0.5 mL of 0.8% methylcellulose in RPMI. The plates were incubated at 370C, 5% CO2 for 4 and a half days.

For staining of infected cells, cell culture medium containing methylcellulose was removed and cells were fixed with 3.7% formaldehyde for 30 minutes. After removal of formaldehyde, the plates were washed with tap water and permeabilized with 1% Triton X-100 in 1X PBS. After a further wash step, infected cells were stained with antibody 4G2 diluted in 1% FBS / 1X PBS and then incubated with polyclonal goat anti-mouse HRP diluted 1: 2,000 in 1% FBS / PBS. A Trueblue peroxidase substrate (KPL) was used for the color reaction. Focus was manually counted for each well and an EC50 value was calculated using Prism software (3-parameter curve fit).

7.3.3. K562 ADE Assay
6 × 10 4 cells / well K562 cells in RPMI / 10% FCS medium were seeded on 96 well round bottom tissue culture plates and cultured overnight. A serial dilution of the antibody was prepared on a separate round bottom plate and a fixed amount of virus was added. The mixture was incubated at 370C for 30 minutes before adding 50 μL to K562 cells. MOI was set to 1. The culture medium was removed overnight and then the antibody / virus mixture was added. After incubating at 370C for 90 minutes, RPMI and 10% FCS were added and incubated for another two and a half days. The cells were then fixed in Cytofix / Cytoperm solution (Becton Dickinson) and then stained with anti-E antibody 4G2-Alexa647 and anti-NS1-Alexa488. Samples were analyzed with a FACS Verse flow cytometer equipped with a 96-well HTS unit (BD).

Example 8: In vivo test of protective ability of dengue-specific antibodies 3C and 3Cam2 against Zika virus infection
8.1. Effectiveness of therapeutic antibody treatment
Instead, we chose the IFNAR model for Zika because AG129 mice are very susceptible to Zika virus and die from infection much earlier than after DENV infection.

IFNAR mice are deficient in type I IFN receptor (IFNAR) but usually express type II or IFNγ receptor (IFNGR).

Based on a previous publication [Cell Host Microbe. 2016; 19 (5): 720-30. Doi: 10.1016 / j.chom. 2016.03.010.], 104pfu / mouse viral load was selected. The deer strain used was isolated in Canada from travelers returning from Thailand. This strain is of Asian strain. Mice were treated with 100 μg doses of antibody.

Mice treated with 3Cam2-LALA + KAES + ACT5 2 days after deer infection showed significantly lower viremia on days 3 and 5 after infection compared to control mice treated with P1 buffer. 3Cam2-LALA + KAES + ACT5 reduced viremia more efficiently than 3C-LALA + KAES + ACT5 (Fig. 16). Survival analysis showed that both antibody-treated groups survived at least 50 days (end of experiment), in contrast to P1-treated mice, which all died by 15 days post-infection (Fig. 17).

8.2. In vivo method
8.2.1. Antibody preparation for in vivo treatment Antibodies are concentrated in P1 buffer (20 mM His, 150 mM Arg-Asp, pH 6.0) at a few milligrams / mL and required in P1 buffer immediately prior to in vivo treatment. Diluted to concentration.

8.2.2. Mice As shown, AG129 or IFNAR mice were used in in vivo experiments. The AG129 mouse was purchased from B & K Universal in the United Kingdom. IFNAR mice (B6.129S2-Ifnar1tm1Agt / Mmjax) were purchased from Jackson Laboratory (USA).

8.2.3. Treatment Treatment Mice were intraperitoneally (ip) infected with 104 pfu / mouse. Forty-eight hours after infection, mice were anesthetized with isoflurane for intravenous (iv) injection of the antibody. Ab was diluted with P1 buffer and injected post-orbit in 100 μL volume.

Example 9: In vitro efficacy of 3Cam2-LALA + KAES + ACT5 against Zika virus
9.1 Join
Binding of DG_3CH-SG182 / 3CL_SK1 (or 3C), DG_3CH-SG192 / 3CL_SK1 (or 3C-LALA) and 3Cam2-LALA + KAES + ACT5 human antibodies to Zika virus (ZIKV) was measured.

Briefly, mAbs were diluted and then incubated on an ELISA plate coated with purified ZIKV at 37 ° C. for 1 hour. HRP-labeled secondary anti-human IgG antibody was used to detect binding of 3C, 3C-LALA and 3Cam2-LALA + KAES + ACT5 to ZIKV virions. Color development was performed using a TMB substrate. After stopping the reaction, the absorbance was measured and readings were performed in pairs. 3Cam2-LALA + KAES + ACT5 binds to ZIKV in the same manner as 3C and 3C-LALA antibodies (Fig. 18).

9.2 The ability of the antibody to suppress ZIKV to infect neutralized cells was also assayed. ZIKV was mixed with diluted human mAb and MOI 10 and incubated at 37 ° C. for 2 hours with gentle stirring. The virus / mAb mixture was then added to HEK 293T cells seeded on 96-well plates. After culturing at 37 ° C. for 2 days, cell infection was measured by detecting ZIKV-specific antigens using flow cytometry. All readings were performed in triads and the neutralizing ability was calculated for cells infected with ZIKV in the absence of antibody. All antibodies, 3Cam2-LALA + KAES + ACT5, 3C and 3C-LALA, show similar ability to neutralize HEK cell infection by ZIKV (Fig. 19).

Example 10: In vivo Efficacy of 3Cam2-LALA + KAES + ACT5 Against Zika Virus: Mother-to-Fetal Transmission The antibody was then tested in vivo to examine the ability of the antibody to block the transmission of the virus from mother to offspring.

Pregnant IFNαR knockout mice were inoculated with ZIKV 10.5 days after mating. Antibodies were administered 1 day, 0 days, and 3 days after infection. On the 16.5 day after mating, pregnant mice were selected and foets were collected. Both whole-body and head fetal weights were recorded; viral load was measured in amniotic fluid, placenta, fetal brain and liver.

Foets from treated mothers weigh significantly heavier than those from untreated mice, which is due to congenital dysgenesis as observed in untreated ZIKV-infected animals. It suggests defense (Fig. 20). Antibodies significantly block the transmission of the virus from the pregnant mother to the foetation (Fig. 21).

Claims (11)

A method for treating or preventing Zika virus infection, which comprises the step of administering an antibody that binds to Zika virus.
(a) (i) Amino acid sequence:

HVR-H3 containing, X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L (SEQ ID NO: 42), HVR-H3,
(ii) Amino acid sequence: HVR-L3 containing _{QQFX 1} X _{2 LPIT, where X 1} is D, S or E and X ₂ is D or A (SEQ ID NO: 45), HVR-L3 ,and
(iii) Amino acid sequence:

HVR-H2 containing, X ₁ is T or R, X ₂ is A or R (SEQ ID NO: 41), HVR-H2;
(b) (i) Amino acid sequence: HVR-H 1 containing _{SX 1} YX _{2 H, where X 1} is N or Y and X ₂ is I or M (SEQ ID NO: 40), HVR- H1,
(ii) Amino acid sequence:

HVR-H2 containing, X ₁ is T or R, X ₂ is A or R (SEQ ID NO: 41), HVR-H2, and
(iii) Amino acid sequence:

HVR-H3 containing, X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L (SEQ ID NO: 42), HVR-H3;
(c) (i) Amino acid sequence: HVR-H 1 containing _{SX 1} YX _{2 H, where X 1} is N or Y and X ₂ is I or M, (SEQ ID NO: 40), HVR -H1,
(ii) Amino acid sequence:

HVR-H2 containing, X ₁ is T or R, X ₂ is A or R (SEQ ID NO: 41), HVR-H2,
(iii) Amino acid sequence:

HVR-H3 containing, X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L (SEQ ID NO: 42), HVR-H3,
(iv) Amino acid sequence:

HVR-L1 containing, where X ₁ is D or E and X ₂ is K or Q (SEQ ID NO: 43), HVR-L1,
(v) Amino acid sequence: HVR-L2 containing _{DASX 1} LKX _{2 , where X 1} is N or E and X ₂ is T or F (SEQ ID NO: 44), HVR-L2, and
(vi) Amino acid sequence: HVR-L3 containing _{QQFX 1} X _{2 LPIT, where X 1} is D, S or E and X ₂ is D or A (SEQ ID NO: 45), HVR-L3 ;or
(d) (i) Amino acid sequence:

HVR-L1 containing, where X ₁ is D or E and X ₂ is K or Q (SEQ ID NO: 43), HVR-L1,
(ii) Amino acid sequence: HVR-L2 containing _{DASX 1} LKX _{2 , where X 1} is N or E and X ₂ is T or F (SEQ ID NO: 44), HVR-L2, and
(iii) Amino acid sequence: HVR-L3 containing _{QQFX 1} X _{2 LPIT, where X 1} is D, S or E and X ₂ is D or A (SEQ ID NO: 45), HVR-L3
Including
Here, the antibody is HVR-H1 containing the amino acid sequence of (i) SEQ ID NO: 11, HVR-H2 containing the amino acid sequence of (ii) SEQ ID NO: 13, and (iii) SEQ ID NO: 16. HVR-H3 containing an amino acid sequence, HVR-L1 containing an amino acid sequence of (iv) SEQ ID NO: 21, HVR-L2 containing an amino acid sequence of (v) SEQ ID NO: 24, and (vi) SEQ ID NO: A method that is not an antibody containing HVR-L3 containing the 27 amino acid sequence. The antibody is a heavy chain variable domain framework FR1 containing the amino acid sequence of SEQ ID NO: 31, FR2 containing the amino acid sequence of SEQ ID NO: 32, FR3 containing the amino acid sequence of SEQ ID NO: 33 or 34, SEQ ID. NO: The method of claim 1 (b), further comprising FR4 comprising the amino acid sequence of 35. The antibody is a light chain variable domain framework FR1 containing the amino acid sequence of SEQ ID NO: 36, FR2 containing the amino acid sequence of SEQ ID NO: 37, FR3 containing the amino acid sequence of SEQ ID NO: 38, SEQ ID NO: The method of claim 1 (d), further comprising FR4 comprising 39 amino acid sequences. A method for treating or preventing dicavirus infection, which comprises the step of administering an antibody, wherein the antibody is at least 95% of the amino acid sequence of any one of (a) SEQ ID NO: 2-6. VH sequence with sequence identity of; (b) SEQ ID NO: VL sequence with at least 95% sequence identity to any one amino acid sequence of 8-10; or (c) SEQ ID NO: 2 A method comprising any one VH sequence of ~ 6 and a VL sequence of any one of SEQ ID NO: 8-10. A method for treating or preventing dicavirus infection, comprising administering an antibody that binds to dicavirus, wherein the antibody comprises (a) an amino acid sequence of SEQ ID NO: 12 HVR-H1, ( b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 15, (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 20, (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, A method comprising (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. The antibody further comprises a polypeptide and
The polypeptide comprises a mutant Fc region containing at least one amino acid modification in the parent Fc region.
The mutant Fc region has a substantially reduced FcγR binding activity and does not have a substantially reduced C1q binding activity when compared to the parent Fc region.
The method according to any one of claims 1 to 5. The mutant Fc region has Ala at position 234, Ala at position 235, and further amino acid modification of any one of (a)-(c) below, according to EU numbering:
(a) 267, 268, and 324;
(b) 236, 267, 268, 324, and 332; and
(c) The method of claim 6, comprising positions 326 and 333. Mutant Fc region according to EU numbering
(a) Glu in 267th place;
(b) Phe in 268th place;
(c) Thr of 324th place;
(d) Ala in 236th place;
(e) Glu in 332nd place;
(f) Ala, Asp, Glu, Met, or Trp at position 326; and
(g) 333rd Ser
The method of claim 7, comprising an amino acid selected from the group consisting of. Mutant Fc region according to EU numbering
(a) Ala in 434th place;
(b) Ala at 434, Thr at 436, Arg at 438, and Glu at 440;
(c) Leu at 428, Ala at 434, Thr at 436, Arg at 438, and Glu at 440;
(d) Leu at 428, Ala at 434, Arg at 438, and Glu at 440.
The method according to any one of claims 6 to 8, further comprising an amino acid selected from the group consisting of. The method of any one of claims 6-9, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 51-59. The antibody
3CH1047 (SEQ ID NO: 6) and 3CH1047 (SEQ ID NO: 6) and having a human IgG1 CH sequence selected from the group consisting of SG182 (SEQ ID NO: 46), SG1095 (SEQ ID NO: 54), and SG1106 (SEQ ID NO: 59). A VH mutant sequence selected from the group consisting of 3CH1049 (SEQ ID NO: 95); and 3CL (SEQ ID NO: 7) and 3CL633 (SEQ ID NO: 60) having the human CL sequence SK1 (SEQ ID NO: 60). 98) The method according to any one of claims 1 to 10, comprising a VL mutant sequence selected from the group consisting of 98).

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